Download In Vitro, Propagation of Strawberry (Fragaria × annanasa Duch

The Islamic University - Gaza
Faculty of Science
Master of Biological Sciences
In Vitro, Propagation of Strawberry
(Fragaria × annanasa Duch.) Through
Organogenesis via Runner Tips
Prepared By
(PDG Y. Youssief
Supervised By
Dr. Abboud El kichaoui
Dr. Tarek El Bashiti
Submitted in partial fulfillment of the requirements for the degree of
master of Biological Science - Botany / Mycology
June / 2009
DECLARATION
“I hereby declare that this submission is my own work and that, to the
best of my knowledge and belief, it contains no material previously
published or written by another person nor material which to a
substantial extent has been accepted for the award of any other degree of
the university or other institute, except where due acknowledgment has
been mad in the text.
Emad Y. Youssief
All Rights reserved: No part of this work can be copied, translated or stored in
retrieval system, without prior permission of the author.
Copy right © by Emad Y. Youssief 2009
I
DEDICATION
I dedicate this thesis to my parents, my wife, and to Youssief, Ahmed and
Abed El Rahman, who always supported my career and helped me to
achieve this degree.
II
In Vitro, Propagation of Strawberry (Fragaria × annanasa Duch.)
Through Organogenesis via Runner Tips
ABSTRACT
Using in vitro tissue culture technique through organogenesis, recognizing the most
important problems during planting stages, and treating these problems. As a result of this,
plants in large numbers and with high quality is produced. Furthermore, this technique can
be applied on other kinds of plants. Growing strawberry with this technique is
economically advantageous.
The cultivated strawberry (Fragaria × ananassa Duch.), a member of the Rosaceae, is the
most important soft fruit worldwide. In vitro techniques are important for clonal
multiplication. An efficient method for sterilization, browning, shoot regeneration and root
formation from runner tips of strawberry plantlets cv. Sweet Charlie was developed.
Runner tips of 1-2 cm long were used as source of explants. In sterilization method, when
the runner tips were immersed in sodium hypochlorite solution (1.5%) containing two to
three drops of Tween 20 per 100 ml for 20 minutes and 0.1% HgCL2 for 10 minutes gave
the maximum aseptic cultures in august, however, in October all the samples were failed
to reduce explantscontamination.
After surface sterilization apical meristems of 3-5 mm long were isolated and used as
explants.
To study the effect of various pretreatments on explants browning and survival percentage,
different sets of sterilized explants were dipped in three different concentrations of
antioxidant solution (1, 1.5 and 2% PVPP) for 1-2 h prior to inoculation with addition of
(0.2g/100 ml) PVPP in the MS free hormone medium and subjecting cultures to an initial
ten days
cold treatment before transfer the explants to growth chamber, the results
indicated that percentage of survival explants when treatment with 1% and 2% PVPP were
20 and 60% respectively in August season.
III
However, when repeated the same experiment in October season, with addition to another
set dipped in sterilized distilled water without antioxidant solution as a control, the lowest
percentage of browning were observed in the control and 2% PVPP were 100 and 70 %
respectively.
At multiplication stage results indicated that the highest auxiliary buds were observed
when MS medium supplemented with 1 mg / l BA.
At rooting stage, it was clear visually
that MS medium supplemented with 0.5 g/l
activated charcoal and MS medium at full strength gave better results in enhanced the root
number and length and shoot number and length per plantlet.
Effect of IBA on the root response, the results indicated that IBA with (0.5, 1.0, 1.5 mg/l)
observed the lowest root response compared to all other treatments.
Because Israeli air force bombed the lab building at the Islamic university in 27-12-2008.
We wrote down data visually in rooting stage.
This research paper is considered the first published in Gaza strip.
Keywords: Propagation, Strawberry, Fragaria spp., Tissue culture techniques,
Benzylaminoburine (BA), Polyvinylpyrollidone (PVPP), Activated charcoal (AC).
IV
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VI
ACKNOWLEDGEMENT
I thank to Allah whose blessings have enabled me to accomplish my thesis work
successfully.
I take this opportunity to thank Dr. Abboud El Kichaoi and Dr. Tarek El Bashiti,
Department of Biology and Biotechnology, chairmen of the supervisory committee, for
their supports, encouragements, and guidance. No words can express my sincere and deep
sense of reverence for them.
I wish to express my gratefulness to all the moral support during my project work.
My sincere thanks to Egyptian Prof. Hamdy Emarah, Genetic Engineering and
Biotechnology Research Institute, Minufiya University, Sadat City, for his constant
support and encouragement during this work.
I would like to thank Prof. Ismail Abdel-Azez and Dr. Abdallah Abed who taught me in
the thesis-related courses.
I am highly thankful to my lab. Staff, Youssief Abu Selmeya, Ashraf Al Shafei and
Mohammed Ouda for their help during my project course.
The whole contribution for my achievements goes to my parents, Brother and Sisters, who
always stood by me during thick and thin, and providing me the moral support and
resources to finish my work. Their unwavering faith in me has been a source of constant
inspiration for me.
VII
CONTENTS
DEDICATION…………………………………………………….…….....…
I
DECLARATION ……………………………………………..…….………..
II
ENGLISH ABSTRACT ………………………………………….….……...
III
ARABIC ABSTRACT ………………………………………….……….…..
V
ACKNOWLEDGMENTS …………………………………………………..
VII
CONTENTS ………………………………………………….........................
VIII
LIST OF TABLES ……………………………………………..……………
XII
LIST OF FIGURES………………………………………….………………
XIII
ABBREVIATIONS ……………………………….…………………………
XIV
CHAPTERS 1
…………………………………………………..….………..
1
1 INTRODUCTION ……………………………………………..………….
1
1.1 Cell Culture ……………………………………………………………...
2
1.2 Application of Tissue Culture ………………………………………….
……………………………………………………
1.3 Stages of the Tissue Culture Method ………………………………….
………………………………….…………
1.3.1 Stage I. Establishment of the Aseptic Culture ……………………….
5
1.3.2 Stage II. Multiplication of Propagula ……………………………...…
6
1.3.3 Stage III. Preparation for Reestablishment of Plants in Soil ……….
6
1.4 Micropropagation Methods …………………………………………….
7
1.4.1 Organogenesis ………………………………………………………….
7
1.4.1.1 Multiplication by Apical and Axillary Shoots……………………...
……………………….
1.4.1.2 Multiplication by Adventitious Shoots……………………………...
……………………………….
1.4.2 Initiation of Callus ………………………………………………...…..
8
1.4.3 Zygotic and Somatic Embryo Development ………………...……….
9
1.4.4 Cell Suspension Culture ……………………………………..……….
10
1.5 Background on Strawberry Farming in Gaza Strip ………………….
………………………………….….
1.5.1 The Most Commonly Grown Strawberry Cultivars in Gaza Strip
…………………….………
1.5.2 Problems Faced Planting Strawberry in Palestine ………………….
10
1.5.3 Impact of the Closure on Gaza Strip Exports of Strawberries …….
13
VIII
6
6
8
8
12
12
1.6 Significance………………………………………………………………
……..………..
1.7 Aim of the Study …………………………………………………………
………………………..
1.7.1 Objectives ……………………………………………………………..
13
CHAPTER 2
…………………………………………………………………
15
2. LITERATURE REVIEW ……………………………………...…………
15
2.1 Palestine Climatic and Agriculture …………………………………….
16
2.2 Strawberry ……………………………………………………………….
16
2.2.1 Taxonomy and Origin of Strawberry ………………………………..
16
2.2.2 Description of Fragaria x ananassa Duch. …………………………..
18
2.2.3 Histological Characteristics of Strawberry ………………………….
19
2.2.4 Strawberry Nutritional Value ………………………………………...
19
2.2.5 Strawberry Phytochemicals and Human Health ………………….
20
2.2.6 Anticancer Effects …………………………………………………
21
2.2.7 Anthocyanins…………………………………………………………
21
2.2.8 The Strawberry Economic in the World ……………………………..
22
2.2.9 Strawberry Diseases …………………………………………...………
23
2.3 Common Problems and Prospect in Tissue Culture …………………..
24
2.3.1 Contamination …………………………………………………………
. …………
2.3.2 Phenolic Browning / Oxidation in Vitro ……………………………...
24
2.3.3 The Difficulty in using Explants from Mature Trees ……………….
28
2.3.4 High – Temperature Degradation of Media Components ………….
29
2.3.5 Hyperhydricity ………………………………………………………..
30
2.3.6 Shoot Tip Necrosis ……………………………………………………
31
2.3.7 Light Requirements …………………………………………………..
33
2.3.8 The Cost of Generating Plantlets …………………………………….
33
2.3.9 Somaclonal Variation …………………………………………………
33
2.3.10 Acclimatization of Tissue-Cultured Plants …………………………
34
2.4 Use of Charcoal in in Vitro Culture …………………………………….
36
2.4.1 Providing a dark Environment ……………………………………….
37
IX
14
14
26
2.4.2 Adsorption of Inhibitory Substances in in Vitro Cultures
37
2.4.3 Adsorption of Plant Growth Regulators in in Vitro Culture ……….
38
2.5 Strawberry Tissue Culture ……………………………………………..
39
2.5.1 Strawberry Organogenesis ………………………………………….. .
…………………………………
2.5.1.1 Strawberry Direct Organogenesis ………………………………….
41
2.5.1.2 Adventitious Shoot Regeneration …………………………………..
46
2.5.2 Strawberry Indirect Organogenesis …………………………………
48
2.5.3 Strawberry Anther Culture and Haploid Recovery ………………...
50
2.5.4 Strawberry Somatic Embryogenesis …………………………………
51
2.5.5 Strawberry Somaclonal Variation ……………………………………
51
CHAPTER 3
54
3 Materials and Methods ……………………………………………………
54
3.1 Materials …………………………………………………………………
55
3.1.1 Source of Explants …………………………………………………….
55
3.1.2 Chemicals……………………………………………………………….
55
3.1.3 Equipments…………………………………………………….….……
56
3.2 Methods………………………………………………………………….
56
3.2.1 Surface Sterilization of Explants…………………………………….
56
3.2.2 Culture Medium ……………………………………………….………
57
3.2.3 Sterilization of the Nutrient Media ………………………………….
58
3.3 Strawberry Micro-propagation ………………………...………………
58
3.3.1 Effect of Pretreatments on Explant Browning………………...……..
58
3.3.2 Multiplication Stage ……………………………………..……………
58
3.3.3 Rooting Stage …………………………………………………………..
59
3.3.3.1 Effect of Different Strengths of MS Medium ………..…………….
59
3.3.3.2 Effect of Different Concentrations of IBA ……………...………….
59
3.3.3.3 Effect of Different Concentrations of Activated Charcoal ….……
59
3.4 Statistical Analysis ………………………………………………………
59
X
41
CHAPTER 4
60
4. RESULTS AND DISCUSSION ……………………………..…………..
60
4.1 Source of Explant ………………………………………………..………
61
4.2 Surface Sterilization of Explants ……………………………….……...
61
4.3 Effect of Pretreatment on Explant Browning ………………….………
64
4.4 Multiplication Stage ………………………..……………………………
68
4.5 Rooting Stage ………………………………………………..…………...
70
4.5.1 Effect of Different Strengths of MS Medium …………….………….
70
4.5.2 Effect of Different Concentrations of Activated Charcoal …….……
71
4.5.3 Effect of Different Concentrations of IBA …………………….…….
71
CHAPTER 5
……………………………………………………...………….
75
5. CONCLUSIONS AND RECOMMENDATIONS ……………...……….
75
5.1. Conclusion ………………………………………………………...……..
76
5.2. Recommendations……………………………………………….………
79
REFERENCES ……………………………………………………..……….
80
XI
LIST OF TABLES
Title
Page No.
Table 1: Following timetable shows further details related to the
11
years where strawberry is planted and the amount of production in
Gaza strip.
Table 2: Nutritional values for 100 grams edible portion of
strawberry.
Table 3: Anatomical and histological differences between roots,
20
36
stems and leaves of plants in vitro and after transplanting ex vitro.
Table 4: A list of the chemicals and reagents used in this work.
55
Table 5: A list of the main equipments used in this work.
56
Table 6: Murashige and Skoog’s Medium (1962).
57
Table 7: Surface sterilization treatments for the elimination of
64
bacterial contaminants in in vitro bud culture of strawberry cv.
Sweet Charlie.
Table 8: The effect of PVPP in the browning inhibition.
65
XII
LIST OF FIGURES
Title
Page No.
Figure 1: 'Sweet Charlie' strawberry plant and fruit.
12
Figure 2: Longitudinal section of (a) axillary shoot apex and
19
(b) stipular shoot apex of strawberry cv, Elsanta.
Figure 3: (A) Swilling of runner tips explants of cv. Sweet Charlie
on MS medium free hormone after 8 days of culture.
Figure 3: (B) Development of bud from runner tips explants after
15 days of culture.
Figure 4: (A) Runner tips explants of strawberry in MS media free
hormone after 4 weeks culture the leaf reach to 2-3 cm.
Figure 4: (B) Explants after 6 weeks in MS media supplemented
with 1.0 mg/l BA.
Figure 5: Explants in multiplication media after 8 weeks, the
axillary buds invades the base of the petioles of the oldest leaves
(first division).
Figure 6: Induction of multiple shoots from Runner explants on MS
medium supplemented with 1.0 mg/L BAP, after 12 weeks of culture.
Figure 7: The effect of different strengths of MS, 1/8, 1/4, 1/2, and
full strength of MS medium on rooting response after 4 weeks.
XIII
67
67
67
67
68
68
71
ABBREVIATIONS
Abbreviation
Full form
AC
Activated charcoal
BA
N6 Benzylaminopurine
BM
Basal medium
CH
Casein hydrolysate
CV
Cultivar
°C
Degree Celcius (Temperature)
2,4-D
2,4-Dichlorophenoxyacetic acid
GA3
Gibberelic acid
2-iP
6-(g,g-dimethylallylamino)-purine or
N6-(2-isopentyl)-adenine
IAA
Indolyl-3-acetic acid
IBA
Indolyl-3-butyric acid
KIN/K
Kinetin, 6-furfurylaminopurine
MS
Murashige and Skoog’s Medium (1962)
ìL
Microlitre
NAA
1-Naphthaleneacetic acid
ppm
Part Per million
PVP
Polyvinylpyrrolidone
RAPD
Random amplified polymorphic DNAs analysis
RFLP
Restriction fragment length polymorphism analysis
TDZ
Thidiazouron, N-phenyl-N'-1,2,3-thidiazol-5ylurea
VAM
Vesicular - arbuscular mycorrhizae
XIV
CHAPTER 1
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1.2 Cell Culture
The technique of plant tissue culture occupies a key role in the second green revolution in
which gene modification and biotechnology are being used to improve crop yield and
quality. Those working with plant cell tissue culture are still concerned with the details of
the technique but are now more involved in its application to fundamental aspects of plant
cell differentiation and development and to the problems of crop improvement [1].
Plant tissue culture is the science or art of growing plant cells, tissues or organs on
artificial media by isolating them from the mother plant [2].
Historically, the science of tissue culture development is linked to the discovery of cell and
subsequent cell theory, which states that the cell is the basic structural unit of all living
things. Plant tissue culture is based on the cell doctrine that states a cell is capable of
autonomy and is potentially totipotent. In 1902, the German botanist Gottlieb Haberlandt
developed the concept of in vitro cell culture. He isolated single cells from palisade tissue
of leaves, pith parenchyma, epidermis and epidermal hair of various plants and cultured on
Knop’s salt solution containing glucose and peptone. In his cultures, cells that synthesized
starch and increased in size survived for several weeks though none of them divided. He
predicted the requirements for cell division under experimental conditions that have been
proved through time. Therefore, Haberlandt is considered as the father of plant tissue
culture. Following Haberlandt, many workers continued working on plant tissue cultures.
In 1939, Gautheret cultivated cambial tissues of carrot root, Nobecourt (carrot), and White
(tobacco) for prolonged periods of time. In strict sense, these were the first true plant tissue
cultures [3].
By using plant tissue culture techniques, complete new plants can be obtained from
different explants through direct or indirect morphogenesis and through somatic
embryogenesis. Direct morphogenesis is the production of shoots from explants without
passing through callus (unorganised tissue) phase while indirect morphogenesis refers to
induction of shoots through callus phase. The culture in which an organised form of
growth can be continuously obtained is referred to as organ culture [2]. The most important
kinds of organ cultures used for micropropagation are meristem cultures, shoot cultures,
embryo cultures and isolated root cultures. Callus cultures, suspension or cell cultures,
protoplast cultures or anther cultures are grouped as unorganised tissue cultures. Although
the basic nutritional requirements of in vitro cultured plant cells are very similar to those
utilized by plants, the nutritional composition varies depending on the type of cells, tissues,
organs, protoplasts and the plant species. There is also a difference in nutritional
requirement among genotypes or cultivars of the same species.
A nutrient medium is defined by its composition of mineral salts, carbon source, vitamins,
plant growth regulators and other organic supplements. A particular medium is identified
by its salt composition unless otherwise specified.
Other additives such as amino acids, vitamins, growth regulators or organic supplements
can be added in any concentrations to a given salt composition to get the desired results.
Although several media have been developed, the MS medium [4] is very widely used in
different plant tissue culture systems. Organic additives and the type and concentrations of
growth regulators that are added to the basic media are generally considered to be the most
important factors that affect the response of plant tissues in culture. However, other
additives such as activated charcoal and polyamines and exogenous factors such as
temperature, light intensity and quality, are important in determining the responses of some
tissues in culture [5]. If cultures are maintained for long time, abnormal phenotypes could
be observed. Most of those changes are physiological and temporary, but some are genetic
and affect the manner in which the plants grow, flower and fruit so that changing the
economic and aesthetic value. Therefore, due to these changes it is important to carefully
evaluate the performance of tissue cultured plants as they grow to maturity in greenhouse,
garden or field [6].
Plant cell and tissue culture are used for clonal propagation, production of disease-free
plants, haploid production, triploid production, in vitro pollination and fertilization,
embryo rescue, somatic hybridization and cybridization, somaclonal and gametoclonal
variant selection, germplasm conservation, secondary metabolite production, and genetic
transformation. Although living cells are considered potentially totipotent, only some cells
that are competent divide and give rise to complete plant in tissue culture. Furthermore, not
all plant species are equally amenable to tissue culture. Although production or
improvement of perennial plants, both woody and herbaceous, using tissue culture
especially for cloning and genetic engineering seem very attractive, the complex seasonal
cycles and life cycles of those plants complicate the control of their growth in tissue
culture [7].
The early establishment of shoot cultures for these perennial plants is one of the important
approaches. Stabilized shoot cultures are excellent sources of cells, tissues and organs that
can be used in further complex procedures such as protoplast generation [8, 9] gene
insertion and transclone recovery. If shoot cultures cannot be readily established, these
advantages cannot be realized. Therefore, a major cause of recalcitrance in perennial plants
is the inability to establish fully stabilized shoot cultures.
It is difficult to generate stabilized shoot cultures for plants that have seasonal growth
dynamics dominated by strong episodic or determinant shoot growth. The relatively slow
growth rate of perennials in culture also complicates the tissue culture procedures as many
perennial tissues release high content of phenolic compounds into a culture medium. In
some cases, some technical approaches can overcome those limitations in tissue culture.
However, development of a deeper understanding of physiological bases of such
genetically predetermined phenomena is important [7].
When Haberlandt 1839 attempted the first plant cell culture, his intentions were to develop
a more versatile tool to explore morphogenesis and to demonstrate totipotentiality of plant
cells. He probably did not suspect that the cell culture technique would become a valuable
aid in economically oriented activitie [10].
The goal of asexual propagation is to produce uniform plants of a selected genotype.
Vegetative propagation usually assures that the desired characteristic of the selected plant
is retained throughout its clone. In contrast, there is no certainty that seedlings will
reproduce even some of the parental characteristics. Nevertheless, seed propagation has
been used in many instances for economic reasons, and major characteristics of a variety
have been reproduced with sufficient consistency e.g. many flowers and vegetables [10].
Asexual multiplication of both hardwood and softwood using tissue culture methods can be
achieved by axillary bud breaking, production of adventitious buds and somatic
embryogenesis.
Plantlets via axillary buds were produced from about 45%
of the
angiosperm, 13% from adventitious buds, 25% indirectly from callus and 17% from
somatic embryogenesis, for gymnosperm, the percentages were 28, 55, 5, and 12%,
respectively. Therefore, for angiosperms the most successful method was axillary bud
multiplication, and for gymnosperms, direct adventitious bud formation [11].
Tissue culture and micropropagation have become important elements in the plant
propagation industry. The total volume of micropropagated plants annually in the world is
estimated to be hundreds of millions of plants. It is reported that around some 50,000
varieties
of plants are propagated in the world using the tissue culture method. The
business of plant propagation involves approximately 600 companies and accounts for
more than 500 million units, the majority of which are ornamental species [12].
1.2 Application of Tissue Culture
During the last 30 years it has become possible to regenerate plantlets from explants
and/or callus from all types of plants. As a result, laboratory-scale micropropagation
protocols are available for a wide range of species and at present micropropagation is the
widest use of plant tissue-culture technology. There were over 300 commercial operators
World-wide in 1990. In Europe, there were 172 micropropagation firms and about 1800
different tissue lines (species and varieties) in culture amongst the 501 plant tissue-culture
laboratories identified in 1993. For example, of the 88 European laboratories using potato
in tissue culture, 58 were listed as using in-vitro multiplication, 49 were involved in the
elimination of pathogens, 45 were using tissue-culture simply to store germplasm, 44 were
involved in genetic modification and 26 had plant-breeding programmes [13].
There are four areas in which applications of plant tissue culture are possible, either
presently or in the near future:
a. production of pharmaceuticals and other natural products;
b. the genetic improvement of crops;
c. The recovery of disease-free clones and preservation of valuable germ plasm; and d.
rapid clonal multiplication of selected varieties. Although there has been substantial
research on the use of cell and organ cultures as sources of pharmaceuticals, a
successful commercial application remains unrealized [10].
1.3 Stages of the Tissue Culture Method
While it has been recognized that the propagation of a plant through tissue culture must
proceed through a sequence of steps, it has not been the general practice to systematically
explore specific requirements of each step. As a commercial procedure, the sequential
steps must be identified and virtually optimum conditions of each should be established. In
principle, there are three major steps, hereafter referred to as stages for the purpose of
emphasis, each with a different objective and possibly different requirements [10].
1.3.1 Stage I: Establishment of the Aseptic Culture
The objective of Stage I is simply to attain an aseptic tissue culture of the plant in question.
The culture may result as enlarging shoot tips, rooted shoot tips, callus, etc. It is only
necessary that the culture be free from obvious infection, that a suitable proportion of
explants survive culture, and that there is rapid growth among the explants [10].
1.3.2 Stage II: Multiplication of Propagula
In Stage II a rapid increase of organs and other structures which can ultimately give rise to
plants is the aim. The increase can be achieved in most instances by either inducing
adventitious organ or embryo formation or by enhancing axillary shoot initiation. With
most species the more likely method of multiplication would be adventitious
organogenesis, either of shoots or of asexual embryos. In many cases an intermediary
callus has been involved. The method of adventitious organogenesis may enable a
substantially faster increase in propagula, it can have serious disadvantages. For example, a
high incidence of genetically aberrant plants has not been uncommon with this method.
The method of axillary shoot multiplication may be slower, but genetically deviant plants
have been virtually absent when it has been used, e.g. Asparagus officinalis and Gerbera
jamesonii [10].
1.3.3 Stage III: Preparation for Reestablishment of Plants in Soil
A successful tissue culture method of propagation must result in reestablishment in soil of
a high frequency of the tissue culture derived plants. Stage III is intended to prepare the
propagula for their successful transfer to soil. Research on Stage III has been largely
neglected. This stage involves the rooting of shoot cuttings, hardening of plants to impart
some tolerance to moisture stress, conferring of a degree of resistance to certain pathogens,
and conversion of plants from the heterotrophic to the autotrophic state. With certain
plants, e.g. bulbs and others adapted to the temperate climate, it may be necessary to
satisfy dormancy-associated requirements.
Bulbs, corms, and tubers may undergo dormancy when transferred from test tube to soil
without having fulfilled their chilling requirements.
Common conditions with respect to nutrient medium and culture environment may
sometimes be applicable to two stages. Thus, it has been possible to proceed through
Stages I and II by utilizing nutrient media of the same composition and form and the same
light and temperature provisions. For maximum success stage III conditions should be
distinct from the other two [10].
1.4 Micropropagation Methods
1.4.1 Organogenesis
Organogenesis referred to the process whereby explants, tissue or cell can be induced to
form root and shoot and even whole plantlets [14].
Embryos are not classified as organs because these structures have an independent
existence that is embryos do not have vascular connections with the parent plant body [15].
Organogenesis in vitro can follow either of the two paths:
Indirect organogenesis by Primary explant is induced to form callus prior to undergoing de
novo organogenesis. The resultant plants may show variations due to the involvement of
the callus phase and direct organogenesis without passing through callus phase is involved
and the regenerants may be identical to the parent plant [16].
The key feature of de novo organogenesis is the formation of a 'meristemoid' arising from
vacuolated parenchyma cells. The cells of the meristemoid are small, isodiametric, and
thin-walled with prominent nuclei and with extensive network of plasmodesmata. This is
the first step of ‘Cellular Differentiation’. It is followed by ‘Acquisition of Competence’,
‘Cellular Determination’ and finally ‘Cellular Differentiation’ giving rise to organs [16].
Organogenesis relies on the production of organs, either directly from an explant or from a
callus culture. There are three methods of plant regeneration via organogenesis:
The firest two methods depends on adventitious organs arising either from a callus culture
or directly from an explant, alternatively, axillary bud formation and growth can also be
used to regenerate whole plants from some types of tissue culture [17].
1.4.1.1 Multiplication by Apical and Axillary Shoots
Micropragation through apical and axillary shoot proliferation is the most reliable
technique for mass multiplication since it ensures genetic stability of clones. Apical and
axillary shoots contain active meristems. Shoot tips cultured on basal medium containing
no growth hormones typically develop into single seedling like shoot with strong apical
dominance. On the contrary when the shoots of the same explant material are grown on
culture medium containing cytokinins or other growth adjuvants, axillary shoots develop
clusters of secondary and tertiary shoots. These clusters can be further subdivided into
smaller clumps of shoots or separate shoots which, in turn, will form similar clusters when
subcultured on a fresh medium. This subdivision process may continue indefinitely
provided the basic nutrient formulations are adequate for normal growth [18].
1.4.1.2 Multiplication by Adventitious Shoots
Adventitious shoots arise naturally on plant tissues located in sites other than at the normal
leaf axil regions. Many ornamental and horticultural species have been successfully
propagated in vitro by adventitious shoot initiation. New adventitious shoots can develop
directly from the explants like root, stem, petiole, leaf lamina, flower parts or indirectly
from the callus cultures obtained from these explants. The initiation of adventitious shoots
is dependent on two factors a) choice of explant; b) hormone regime to which plant is
subjected [18].
1.4.2 Initiation of Callus
A callus consists of an amorphous mass of loosely arranged thin walled parenchyma cells
arising from the proliferating cells of the cultured explants. Frequently, as a result of
wounding, a callus is formed at the cut end of a stem or root. Using tissue culture
techniques, callus formation can be induced in numerous plant tissue and organs that do
not usually develop callus in response to an injury. Plant material typically cultured
includes vascular cambia, storage parenchyma, pericycle of roots cotyledons, leaf
mesophyll, and pro-vascular tissue. In fact, all multicellular plants are potential sources of
explants for callus initiation [15].
Many parts of a whole plant may have an ultimate potential to proliferate in vitro, but it is
frequently found that callus cultures are more easily established from some organs than
others. Young meristematic tissues are most suitable, but meristematic areas in older parts
of a plant, such as the cambium, can give rise to callus. The callus formed on an original
explant is called ‘primary callus’. Secondary callus cultures are initiated from pieces of
tissue dissected from primary callus. Subculture can then often be continued over many
years, but the longer callus is maintained, the greater is the risk that the cells thereof will
suffer genetic change [19].
1.4.3 Zygotic and Somatic Embryo Development
Somatic embryos may be developed from different vegetative tissues. The term somatic is
an adjective meaning “of the body”, but is broadly applied to dividing (living) tissue.
Embryogenesis refers to the generation of embryos. Somatic embryogenesis may then be
interpreted as the generation of embryos from plant organs or tissue. In common
vernacular, somatic embryogenesis is the “cloning” of embryos and refers to dividing
tissue. After the initial stages, somatic embryos develop through a sequence of stages that
resemble those observed for developing zygotic embryos (embryos derived from sexual
reproduction) [20].
In theory, embryogenic tissue may be developed from any plant explant. An explant may
be a mature plant organ, such as a root, stem, leaf or bud, or it may be a cell or group of
cells. In practice, tissues from the cotyledon, the meristem, or the zygotic embryo are
preferred.
Somatic embryogenesis (SE) is a tissue culture approach where proliferative embryo
suspensor masses are established from non-meristematic cells and subsequently cultured to
produce organized bipolar structures possessing shoot and root meristems (that is, somatic
embryos) [20].
Somatic embryogenesis can be initiated directly from "preembryonic determined cells" that
are programmed for embryonic differentiation, where plants are genetically identical
(clonation) or indirectly from unorganized tissues (callus) and embryos originate from
"induced embryogenic cells" within the callus. Propagation by indirect embryogenesis
carries the risk of producing plants that may differ genetically from each other and from
the parental plant [21].
1.4.4 Cell Suspension Culture
A suspension culture originates with a "random critical event "occurring during the early
exposure of the plant cells to the liquid medium. Cells undergoing this transition in
metabolism and growth rate produce a "cell line". Cell suspension cultures are generally
initiated by transferring fragments of undifferentiated callus to a liquid medium, which is
then agitated during the culture period. Although a longer time is required, suspension
cultures can be started by inoculating the liquid medium with explants of differentiated
plant material (e. g, a fragment of hypocotyls or cotyledon) [15].
The growth of plant cells is more rapid in suspension than in callus culture and is also
more readily controlled because the culture medium can be easily amended or changed.
Organs can be induced to develop in cell suspensions: root and shoot initiation usually
commences in cell aggregates. Somatic embryos may arise from single cells. Cells from
suspensions can also be plated onto solid media where single cells and/or cell aggregates
grow into callus colonies from which plants can often be regenerated. For these reasons
suspension cultures might be expected to provide a means of very rapid plant
multiplication [19].
1.5 Background on Strawberry Farming in Gaza Strip
Growing strawberry in Palestine started in 1976. The area in which strawberry was grown,
1-2 dunums. The growing of strawberry aimed to:
a- Studying how successful is planting strawberry according to climate, soil and
irrigation water.
b- Studying how acceptable is the crop to the farmer.
The situation continued as it as was tell 1971. At that time, planting strawberry was mainly
planted in the north Gaza where water (less than 250 ppm Chloride) and soft sandy soil are
available. The crop continued to be planted in the north of Gaza till the arrival of the
Palestinian authority in Gaza Strip, 1993. After the arrival of Palestinian authority, nearly
30 dunums were planted with strawberry in Rafah and Khan Yunus, especially in AlMawasi area. The thing continued for two years. However, because of the Israeli actions,
planting strawberry in the Rafah and Khan Yunus stopped. Farmers were unable to export
or market their crops [22].
So far, four main organizations and other some private exporters have been responsible for
exporting strawberry abroad. Lately, EURO GAP is used in planting strawberry to secure
high quality in all the processes. In addition, environment and saving environments quality
caution procedures are put under consideration. Strawberry is now exported within the
Palestine group called Coral [22].
(Table 1): Following timetable shows further details related to the years where
strawberry is planted and the amount of production in Gaza strip [22]:
No.
Year
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1967-1968
1971-1972
1975-1976
1979-1980
1983-1984
1987-1988
1991-1992
1995-1996
1999-2000
2003-2004
2004-2005
2005-2006
2006-2007
2007-2008
Produce (Tone)
land area (Dunum)
1.5
12
50
900
1300
1400
1500
4098
4800
5757
7500
2500
2200
2350
1
12
25
300
327
358
373
1360
1675
1919
2500
2500
2500
2500
1.5.1 The Most Commonly Grown Strawberry Cultivars in Gaza Strip
At the end of 1960s and the beginning of 1970s following items are planted:
Fresno, Aliso and Tioga.
During 1980s following items are planted:
Douglas, Sharon, Ofera, Dorit, Rosalinda and Shandler.
During 1990s following items are planted:
Osso Grandi, Yael 329, Camarosa, Tamar 328 and Sweet Charlie.
'Sweet Charlie' strawberry (Fragaria x ananassa) offers strawberry growers in Florida, and
other areas with relatively mild winter climates, a short-day cultivar that is early fruiting
and produces fruit that has a distinctively sweet and flavorful due to a consistently low acid
content and is resistant to anthracnose (caused by Colletotrichum acutatum) (Figure 1).
Figure 1: 'Sweet Charlie' strawberry plant and fruit.
1.5.2 Problems Faced Planting Strawberry in Palestine
a-
Inability to grow high quality strawberry mother plants in local greenhouse.
It’s totally relayed on Israeli companies to do that.
b-
Strawberry mother plants which are got from Israeli companies are expensive.
c-
Strawberry is planted in one governorate in Gaza Strip.
d-
Strawberry is exported through Israeli companies since Palestinian companies are not
able to do that.
e-
Continuous closer of the border between Gaza Strip and Israel. As a result, exported
crops stay on the border and it spoils as fungi spread in it.
1.5.3 Impact of the Closure on Gaza Strip Exports of Strawberries
Despite the relatively small area of farmland used for sector, it is considered that enjoy
high economic and social value. Strawberries annual income is estimated at $US 10-12
million. In addition, it contributes to the creation of employment opportunities. Strawberry
farmers are estimated at 450. Indirect employment opportunities related to the Strawberry
sector (production, marketing, services, etc.) are estimated at 7,500 jobs.
The total losses of farmers in the sector of strawberry for last season amounted to $US
11.25 million. Approximately 450 strawberry farmers.
The strawberry sector losses were estimated by the researcher according to official
statistics. The yield of 1 dunum is 2 tons and the dunums that export strawberries are 750
dunums. The price of 1 exported tons 30,000 NIS (an average of $US 7,500). The total loss
is 750 x2 x7500 = 11,250,000 $US [23].
Therefore, building tissue cultures planting unit which is able to produce strawberry
mother plants with high quality and resistance to common fungal and bacterial diseases.
Hence, we, Palestinians will be independent on the Israeli side. Moreover, we will be able
to produce by ourselves with low a price which suits the abilities of our farmers. We will
also be able to choose the item which fit in with the requirements of international
marketing web.
1.6 Significance
Our study is the first in Gaza, so the establishment of a model for plant tissue culture and
determining the essential factors that influence the success of tissue culture technique
present a very important point in the biotechnology field in our country, especially in the
cell culture domain. The choice of strawberry was economically successful in the
agriculture field. It is noticed that strawberry average production between 1983 and 2005
was 3 Tons per dunum. However, average production of strawberry was less than a Ton
per dunum during 2006 – 2008.
Durind 1983- 2005, farmers used to depend on strawberry mothers plants imported from
Israel. On the other hand, average production was less during 2006 – 2008 that farmers had
to use plants they had due to the Israeli closure on Gaza strip.
Above stated facts show the importance of strawberry tissue culture in our country.
1.7 Aim of the Study
In vitro propagation of strawberry (Fragaria × ananassa Duch.) by using runner tips.
1.7.1 Objectives
The main objectives of the present study were:

To test methods of avoiding problems in the beginning of the micropropagation.

To prepare a particular and selective media for culturing Fragaria × ananassa
Duch.

To overcome the problem of oxidation and minimize phenolic exudation.

To find out the effect of some regulators and supplements on the growth of shoots
and roots of this plant.

To refine the reorganization of the strawberry micropropagation stages.
CHAPTER 2
Literature Review
2.1 Palestine Climatic and Agriculture
Gaza Strip lies on the eastern coastal of the Mediterranean Sea. Gaza Strip lies in the
moderate northern area of the horizontal lines 31.13 – 31.36 to the north of the equator
which gives it good a climate along the seasons of the year. The total area of historical
Palestine is 27009 Km2, among which Gaza occupies only 1.35%. According to survey
conducted in 2006 by Palestinian Center Bureau of Statistics (PCBS), 48% of Gaza Strip
land is cultivated; out of which, 32% is cultivated with fruits, 32% is cultivated with
vegetables and 36% is cultivated with field crops [24].
2.2 Strawberry
2.2.1 Taxonomy and Origin of Strawberry
Strawberries are members of the family Rosaceae, subfamily Rosoideae, and genus
Fragaria. Closely related genera include Duchesnea, the mock strawberry, and Potentilla,
the cinequefoils. Fragaria species can be grouped by ploidy: there are nine diploids, two
tetraploids, one hexaploid, and four octoploids. Diploids (2n=14) include F. vesca Duch.,
F. viridis Duch., F. nilgerrensis Schlect., F. daltoniana. J. Gray, F. nubicola Lindl. ex
Lacaita., F. iinumae Makino, F. yezoensis Hara, F. nipponica Makino, and F.
mandschurica Staudt [25].
The alpine strawberry, F. vesca, is the most geographically widespread. Cytogenetic
studies indicate that this species may be a diploid progenitor of the octoploid strawberries.
The two tetraploids (2n=28) are F. orientalis Losinsk and F. moupinensis (Franch.) Card.
The lone hexaploid (2n=42), F. moschata Duch. Or musky strawberry is found in northern
and central Europe into eastern Russia. This species was domesticated in the early 1600’s
and fruit were commonly known as ‘Hautbois’ or ‘Hautboy’. Cultivated plantings still
exist in Europe.
Four octoploids (2n=56) are known: F. iturupensis Staudt, F. chiloensis (L.) Duch., F.
virginiana Duch., and F. ×ananassa Duch. F. iturupensis is found in Iturup Island of the
Kuril Islands (northeast of Japan) [26]. Taxonomic characteristics include obovate subglaucous leaves (similar to F. iinumae), hermaphroditic flowers, and almost spherical fruit.
The beach or Chilean strawberry, F. chiloensis, is found along the Pacific coast from
Alaska down through central California, along the beaches of Chile and inland to the
Andes Mountans, and on top of mountains in Hawaii. They were once extensively
cultivated in Chile, Peru, and possibly Ecuador. Wild populations are primarily dioecious
although hermaphrodites have been found in California [27, 28]. Plants are low-spreading,
vigorous, and produce many runners. Leaves are thick, dark-green and very glossy. Fruit
are dull to bright red, firm, white fleshed, pungent, and large. Four subspecies are
recognized based on morphology and distribution [29]: ssp. chiloensis (South America),
ssp. lucida (Washington to California), ssp. Pacifica (California to Aleutian Islands), and
ssp. sandwicensis (Hawaii).
The scarlet or Virginia strawberry, F. virginiana, is found North America from the
Southeastern U.S. north to Newfoundland and as far west as the Yukon Territory into
Alaska, oftentimes in meadows. Plants are slender, tall, and have many runners. Only
females and hermaphrodites are observed in the eastern U.S. while all three sexes are
equally found in western populations [30, 31]. Fruit are soft, round, up to 1.5 cm diam,
light red, aromatic, with deeply embedded seeds, and white flesh. Both plant and fruit
characters are highly variable. Four subspecies are recognized by (Staudt (1989) [25] ssp.
virginiana Duch. (Eastern U.S. to Newfoundland and west to Yukon Territory); ssp. glauca
(Wats.) Staudt [southern Arizona through the Rocky Mountains into northwest Canada and
central Alaska, probably equivalent to F. ovalis [28] due to lack of hybridization barriers
and intermediate characters]; ssp. platypetala (Rydb.) Staudt (California to British
Columbia and in the Rocky Mountains in Colorado and Wyoming); and ssp. grayana (E.
Vilmorin ex Gay) Staudt (Texas through Louisiana, Alabama and north to New York).
However, this classification scheme by Staudt has undergone considerable debate. New
evidence suggests that these four “subspecies” are too closely related to be considered
infraspecific taxa. Welsh et al. [32] suggested that ssp. glauca and ssp. platypetala
completely intergrade and should be referred to as a single taxa var. glauca. Hokanson et
al. [33] suggested that strawberries in the Black Hills and eastern front ranges of the Rocky
Mountains may be introgressive swarms between ssp. glauca and ssp. grayana. Finally,
Harrison et al. [34] using multivariate analysis, found that these strawberries from the
Black Hills were morphologically intermediate between collections of eastern ssp.
virginiana and western ssp. glauca. Furthermore, when multivariate analysis was done
using RAPD data, this population of strawberries from the Black Hills was part of a large
cluster group that encompassed the eastern ssp. virginiana and western ssp. glauca.
The dessert or pineapple (Ananas) strawberry, F. × ananassa is the most important
cultivated strawberry worldwide. It arose as a chance hybrid of F. chiloensis x F.
virginiana within European gardens during the mid-1700’s. Naturally occurring hybrids
have been found in coastal areas of southwest British Columbia, Washington, Oregon, and
northern California. Staudt (1962) [29] recognized these hybrids as F. ×ananassa nm.
cuneifolia (Nutt. Ex Howell). Many of the dessert strawberry’s traits are intermediate to its
parents. Plants have large fruit, high yields, and vigor.
2.2.2 Description of Fragaria x ananassa Duch.
The F. x ananassa is a perennial which arises from a crown of meristematic tissue or
compressed stem tissue. Leaves, stems, runners, axillary crowns, inflorescences, and roots
all arise from the crown. The plant has trifoliate leaves which spiral around the crown, with
buds in the leaf axils giving rise to the runners. Runners have two nodes with a plant
produced at the distal node. Strawberry blossoms contain many pistils, each with its own
style and stigma attached to the receptacle. When fertilization occurs the receptacle
develops into a fleshy fruit [35].
The fruit is called an achene which contains the seeds. The edible part is an accessory type
fruit. The seeds are arranged on the outside of the receptacle tissue. The growth of the
receptacle is dependent on successful fertilization of the ovules with its size and shape
dependent on the number of achenes formed. Strawberry plants are day length dependent
with cultivars being long day, short day or day neutral [35].
Following further hybridizations, especially since 1850, Fragaria x ananassa has
developed into the large, fragrant, tasty red fruit that is now cultivated worldwide. The
high degree of genetic heterozygosity present in Fragaria spp. enabled the development of
strawberry cultivars adapted to widely varying environment conditions and resistant to
several diseases and pests. Not only the genetic variability, but also a high adaptability and
plasticity of the strawberry plant itself give this crop such a remarkable range of adaptation
[28].
That heterozygosity was explained by (Gaafar and Saker 2006) as there are more than 20
Fragaria species worldwide, there are seven basic types of chromosomes that they all have
in common. However, they exhibit different polyploidy. Some species are diploid, having
two sets of the seven chromosomes (14 chromosomes total). Others are tetraploid (4x =
28), hexaploid (6x = 42), octoploid (8x = 56) or decaploid (10x = 70) [36].
2.2.3 Histological Characteristics of Strawberry
Microscopic observations (Figure 2. a, b) showed that the apex of stipular shoots presented
more sites of higher cell activity in comparison to axillary shoots. In the former, one
axillary meristem was developed at nearly each foliar premordium. Moreover, the number
of primordia on the apical meristem is more important in stipular than in axillary shoots
[37].
Figure 2: Longtudinal section of (a) axillary shoot apex and (b) stipular shoot
apex of strawberry cv, Elsanta. A.m = apical meristem; a.s = axillary shoot; f.p
= foliar primordium. G × 100 [37].
2.2.4 Strawberry Nutritional Value
2.2.4 Strawberry Nutritional Value
For many centuries before, strawberries had been a favorite among the fruits of the
temperate world. They were valued for delicious flavor and fragrance, for health-restoring
qualities and as harbinger of spring [38].
Flavourful and nutritious, strawberries are enjoyed by millions of people in all climates,
including and are predominantly used as fresh fruit. Their use in processed forms such as
cooked and sweetened preserves, jams or jellies and frozen whole berries or sweetened
juice extracts or flavorings, and their use in making a variety of other processed products
made them one of the most popular berry crops, more widely distributed than any other
fruit [39].
Of its many positive characteristics, the nutritional value of strawberries is nearly perfect
(Table 2). Eight medium strawberries contain more vitamin C than an orange, 20% of the
recommended daily allowance for folic acid, no fat, no cholesterol and are considered high
in fiber [40].
Strawberry is cultivated all around the world, not only for its digestive and tonic properties,
but because of the nutritional value of its fruits, important source of folate, vitamin C,
fiber, potassium, flavonoids, autocianidin, phytochemicals and antioxidants.
(Table. 2) Nutritional values for 100 grams edible portion of strawberry (USDA, 1999)
[41]:
Protein
Fat
Fiber
Carbohydrates
Calcium
Iron
Mangnesium
Phosphorus
Potassium
0.61g
0.37mg
2.3g
7.02g
14mg
0.38g
10mg
19mg
166mg
Sodium
Zinc
Copper
Manganese
Selenium
Vitamin B-6
Folate
Vitamin A, IU
Vitamin A, RE
1mg
0.13mg
0.049mg
0.29mg
0.7 µg
0.059mg
17.7µg
3 µg
0.14mg
2.2.5 Strawberry Phytochemicals and Human Health
Although strawberry is not an essential component of the diet, its delicious flavor and taste,
attractive appearance and seasonal availability make this fruit an excellent crop. Even
more, strawberries are rich in phytochemical compounds with potential antioxidant
compounds, mainly ellagic acid and flavonoids, which can lower the risk of cardiovascular
events and tumorogenesis [42].
These qualities have ensured that the economic importance of this crop has increased
throughout the world and, nowadays, it remains as a crop of primary interest for both
research and fruit production [43].
A growing body of data suggests that consumption of a phytochemical-rich diet reduces
the risk of certain chronic human illnesses such as cancer, heart and neurodegenerative
diseases. Strawberry (Fragaria x ananassa Duch.) fruits are a rich source of
phytochemicals (plant chemicals) of which phenolic compounds predominate. Berry fruits
are reported to contain a wide variety of phenolics including hydroxybenzoic and
hydroxycinnamic acid derivatives, anthocyanins, flavonols, flavanols, condensed tannins
(proanthocyanidins) and hydrolyzable tannins [44].
Studies conducted in vitro indicate that berry phenolics have a wide range of biological
properties such as anti-cancer, antioxidant, anti-inflammatory, and cell regulatory effects
[45, 46].
2.2.6 Anticancer Effects
The anticancer effects of individual phytochemical constituents of strawberries, as well as
whole strawberry extracts, have been demonstrated [45]. These anticancer effects are
exerted through multi-mechanistic means of action including the antioxidant actions of the
berry’s phenolic constituents by protecting DNA from damage, and also through effects
exerted beyond antioxidation [46]. The biological activities of strawberry phytochemicals
include the regulation of phase-II enzymes and the modulation of gene expression and subcellular signaling pathways of cell proliferation, angiogenesis and apoptosis (programmed
cell death). Although there have been many published reports on the anticancer effects of
individual phenolics known to be present in the strawberry fruit [47].
Strawberry extracts have also been evaluated for their ability to inhibit mutation by the
direct-acting mutagen methyl methanesulfonate, and the metabolically activated
carcinogen, benzopyrene. Ethanol extracts from freeze-dried fruits of several strawberry
cultivars were also evaluated and hydrolyzable tannin-containing fractions from
strawberries were found to be most effective at inhibiting mutations [48].
2.2.7 Anthocyanins
Anthocyanins are natural pigments providing scarlet to blue colors in flowers, fruits, leaves
and storage organs. The recent interest in the field of anthocyanin chemistry has been
generated by restriction and limitation of the use of synthetic dyes as food ingredients.
Because of low toxicity of anthocyanins, they have a high potential as a food colorant as
the substitute of synthetic red dyes. Recently, these anthocyanins have been thought to
have pharmacological effects, such as lowering the atherogenic index [49] and decreasing
triglyceride and free fatty acid levels [50]. Moreover, Kamei et al. [51] reported that
anthocyanin was more effective to inhibit the growth of tumor cells than other flavonoids.
Studies concerned with anthocyanin production using plant tissue cultures have therefore
become very important.
Masayuki et al. [52] demonstrated that Fragaria ananassa (strawberry) callus, which
produced high amounts of anthocyanin in the dark which accumulated more than 1000 µg
of anthocyanin per g fresh cell. And Sato et al. [53] reported that in the suspension cultures
of F. ananassa cells, anthocyanin content increased with the intensity of light irradiation
from 2500 lx to 8000 lx. They also measured the ratio of pigmented cells in total cells at
8000 lx and found that the ratios were 26% in MS medium.
2.2.8 The Strawberry Economic in the World
Strawberry world production has shown a significant increase in the last 25 years. From
1980–2004, the land cultivated with strawberry increased by 25% and fruit production by
73%. In 2004, strawberry was cultivated on 214,118 ha, reaching a production of 3.1
million tones. Fifty seven countries contributed to this production, though only nine of
them accounted for more than 72% of the total. During the period 2000–2004, average
world production was 3.17 million tonnes, the major strawberry producer nations being the
USA. The total value of USA production was 1.22 billion dollars in 2002, being the third
fruit in value in the non citrus category behind grapes and apples. It is noteworthy that
most strawberry production is localized in areas with mild winter climates [43].
Approximately 15% of world strawberry production is exported as fresh fruit and, at least
in the USA, more than 25% is processed. The major fresh strawberry exporters are Spain
(212,300 t in 2003, 81% of its strawberry production) and the USA (94,600 t, 11.2%).
Spain is the main supplier for the European market during the winter period (February and
March) and Germany, France and the UK are the major consumers of this production. In
Spain, as well as other Mediterranean nations, the concentration of strawberry production
over short periods often creates a product excess, reducing retail prices [54].
In the case of the USA, exported fruits are destined mainly for Canada and a small quantity
for Japan and Mexico. California has more than 85% of the USA strawberry production.
This region also supports the most active and successful strawberry breeding programs. In
fact, the program of the University of California has released very popular varieties (e.g.
Camarosa, Chandler, Seascape, Selva, Pajaro, Oso Grande), accounting for more than 50%
of the world’s strawberry production [43].
2.2.9 Strawberry Diseases
Several soil pathogens damage strawberry roots, resulting in vigor declines and
ultimately death. Two very common problems across the world are red stele or red core
caused by Phytophthora fragariae Hickman and Verticillium wilt caused by Verticillium
albo-atrum Reinke & Berth. And V. dahlia. Black root rot is also widespread and is caused
by a complex of organisms including Pythium, Rhizoctonia and the root lesion nematode
(Pratylenchus penetrans Cobb) [55].
Fusarium wilt or Fusarium yellows (Fusarium oxysporum Schl. f. sp. fragariae Winks and
Williams) is of major importance in Japan, Korea and Australia. Fumigation has been
widely employed to control soil pathogens, but the impending ban on methyl bromide
fumigation has stimulated increased interest in developing resistant cultivars. Without
fumigation, cultivars yield 50% less fruit on average [55].
This chemical is, however, very hazardous to the environment and will be forbidden in
2005 at least in the EU countries. Biological and other solutions are therefore urgently
needed to ensure the health and productivity of strawberry [56].
Among the foliar diseases, three are very widespread and can cause serious damage
including, leaf blight [Phomopsis obscurans (Ell. and Ev.) Suton], Ramularia leaf spot,
[Mycosphaerella fragariae (Tul.) Lindau] and leaf scorch [Diplocarpon earliana (Ell. &
Everh.) Wolf]. Alternaria leaf spot or black leaf spot (Alternaria alternata (FR.) Keissler)
causes serious damage in Europe, New Zealand and Korea [55].
Powdery mildew [Sphaerotheca macularis (Wallr. Ex Fr.) Jaez] is also found across most
of the strawberry range, although it rarely does economic damage. Angular leaf spot,
Xanthomonas fragariae Kennedy and King, is a rapidly growing problem in strawberries
all across the world [55].
Anthracnose is a common problem in strawberries, causing a wide array of symptoms
including fruit rot, crown rot, and lesions of the stolons, petioles and leaves. Anthracnose
diseases of strawberry are caused by Colletotrichum fragariae A. N. Brooks, C. acutatum
J.H. Simmonds, and C. gloeosporioides (Penz.) Penz. & Sacc. In Penz. Colletotrichum
acutatum is the primary pathogen causing crown rot in Europe, while C. fragariae is the
most common cause of crown rot in the southeastern U.S.A. C. acutatum is the primary
pathogen in Israel and California [55].
Phytophthora cactorum (Leb. & Cohn) Schroet also causes widespread incidences of
severe crown (Cactorum crown rot) and fruit rots (leather rot), particularly in warm
climates [55].
2.3 Common Problems and Prospect in Tissue Culture
2.3.1 Contamination
One of the main problems encountered with in vitro propagation was the massive bacterial
contamination at the initiation and multiplication stages. After transfer of the bud on to
solid sterile medium, a whitish exudate of bacteria was observed around the base of the
explant after 2-3 d. The problem was complicated further by the latent nature of the
contaminants. Turbidity of the liquid multiplication medium 1-2 d after splitting of the
shoot clumps indicated the presence of bacteria. In some cases the contaminants appeared
upon the sixth or seventh subculture. Contamination at the initiation stage caused rotting of
the bud whereas at the multiplication stage, the rate of tillering bore an average three- fold
decrease with subsequent death of the plantlets in about one month [57].
In most laboratories, losses due to contamination average between 3-15% of plants at every
subculture [58], rendering commercial micropropagation less economical. It also leads to
severe production losses, low progeny performance and rejections of entire shipments of
plants due to quarantine regulations [59].
Bacterial contamination is one of the most crucial problems of plant tissue culture, both in
research and commercial production. Often bacterial contamination is difficult to detect.
Even healthy plants can contain several bacteria, and some plant exudates may look similar
to bacterial growth. Contaminated plants may lack symptoms, have reduced multiplication
rates, reduced rooting rates, or may die [60].
Contaminants in the xylem vessel which are protected from surface sterilization are
endophytic bacteria detected even in meristem-tip explants .Endophytic bacteria have
probably evolved a close relationship with their host plant through co-evolutionary
processes and may influence plant physiology in ways that have not yet been elucidated
.Inside the plant they have very little microbial competition and usually they do not cause
visible symptoms to the plant. The bacteria may stay latent or symptomless up to several
months after the initiation of culture and may not survive outside the plant tissue
.Endophytic bacteria may even promote beneficial effects for field grown crops, but in
stress conditions such as in vitro culture, latent endophytic bacteria may become
pathogenic and detrimental to the growth and development of the plantlets [61].
Latent bacterial contamination during the proliferation phase adversely affects rooting due
to changes in growth regulators and water potential of the media [62] and also kills young
trees after weaning [63]. Many Bacillus spp. increase pH of the medium, inhibiting proton
pumps involved in plant nutrient uptake and cause precipitation of many nutrients from the
medium [62]. Cultures of apricot with latent bacterial contaminants showed high CO2 and
low O2 concentration in the vessel headspace, thereby decreasing the proliferation and
photosynthetic rates [64].
Organic soil amendments have been observed to increase the inoculum for the
contamination of plants and thus organic material should be avoided in the preparation of
plants for micropropagation. Also, explants taken from different locations have been
observed to differ in their in vitro bacterial contamination. It has been demonstrated that
many of these non fastidious bacterial contaminations are environment associated, and may
be transmitted through water. Bacteria have been detected especially from tissues close to
the soil [65].
The most serious problem for continuos use of antibiotics is the contaminant becoming
resistant, although scientific reports of this problem are scare. Bacteria may be associated
with plant species, which are also sensitive to antibiotics. Antibiotic at bactericidal
concentrations may be toxic to the plant and reduce plant growth. On the other hand, many
antibiotics have enhanced shoot regeneration, shoot development; delayed the loss of
regeneration potential; stimulated callus growth and root formation; and enhanced plant
differentiation from somatic embryos. Additionally, antibiotics may stimulate enzymes
responsible for nitrogen metabolism; Hill reaction in chloroplasts, and increase
chlorophyll, carotene, and xanthophyll content in leaves [61].
Piyarak et al. [60] detected the microbial contaminant in surface-sterilized strawberry
runner explants partially submerged in half strength liquid MS Piyarak medium. 45 of 70
strawberry genotypes were found to be contaminated, there were more bacterial than
fungal contaminants. Bacterial contaminants from 22 strawberry genotypes were isolated,
purified, and identified to genus by standard biochemical tests such as Gram's stain,
motility, oxidase, and gelatinase, and carbon source utilization (Biolog Microplates,
Biolog, Inc., Hayward, CA). Among the 30 isolates identified with the Biolog system, the
majority were fluorescent pseudomonads including Pseudomonas fluorescens types A, F,
and G. P. corrugata, P. tolaasii, P. paucimobilis, Xanthomonas campestris, Xanthomonas
spp., and Enterobacter cloacae were also identified. Five Gram-negative and two Grampositive contaminants could not be identified by the Biolog test. Biochemical tests were
used to characterize the bacteria and to confirm Biolog test results. Bacteria identified in
this study were soil, water, and plant related, indicating that efforts to reduce explant
contaminant levels should be centered on the care of stock plants or the sterility of the
watering system.
2.3.2 Phenolic Browning / Oxidation in Vitro
The presence of phenolic compounds causing death of explants has been another important
problem of tissue cultures of woody perennials [66] in addition to various bacterial and
fungal contaminants as discussed above. Some of these exudates appear as a reaction to
injury and/or infection. In tissue culture they appear after tissue excision and are many
times aggravated by growth media constituents [67]. Tissue blackening occurs due to
action of copper-containing oxidase enzymes: polyphenoloxidases like tyrosinases, which
are released or synthesized in oxidative conditions after tissue wounding and they oxidize
o-diphenols released due to cellular wounding to O-quinones [68, 69]. The onset of tissue
browning has been found to be associated with changes in protein pattern, amino acid
content, ethylene production and the occurrence of saccharose and accumulation of starch
[70]. These changes eventually lead to growth inhibition or death of explants. Other types
of phenolic exudates appear at the end of incubation period and are apparently products of
dying cells [67]. The phenolic exudation is aided by light and is autocatalytic.
It is possible, that some external factors trigger stress symptoms such as browning in plant
tissue. These factors may be pathogens or in some cases even agar. High concentrations of
other medium components such as macrosalts, auxins, and sucrose have caused browning
as well. In addition, there are some substances including 5, 6-Cl2-IAA, yeast extracts, and
phloridzin which directly enhance the production of phenolic compounds [61].
Several factors in the growth and culture conditions of the donor tissues have been tested,
that may in later steps of the establishment of tissue cultures cause browning. In vitro
formation of phenolic acids from explants diminished when donor tissues were
preincubated on a medium with or without mineral salts treated with BA (200 mg\ L) or
PVPP, ascorbic acid, cysteine, or citric acid [61].
Accumulation of phenolics is most obviously associated with the developmental stage of
the plant and season, which was shown with woody species. Explants collected from
November to February had produced low browning percentages in vitro, whereas the
browning was at its maximum if explants were collected in April - August [71].
Differences in browning between varieties and in relation to the size of the explant were
also observed. The authors concluded that a better way to overcome tissue browning was
to optimize growth conditions of the source plants rather than by treating the explants
afterwards. The most effective antibrowning treatment was growing the source plants in
the dark, in the greenhouse, or use of explants from heat treated or cold-treated source
plants [72, 73]. Simple, but effective inhibition was achieved by sealing the cut ends of
explants with paraffin [74].
Many medium components have been observed to decrease or eliminate tissue browning
such as inclusion of nitrate as a source of nitrogen; increased concentration of phytagel or
using gelrite instead of agar. Many authors have also tested phenol traps such as activated
charcoal; adsorbent resin; citric acid; cysteine; PVPP; and antioxidants such as ascorbic
acid or glutathione (GSH). In addition, culturing in vitro plantlets under low light intensity
has given good results [61].
The initial browning develops later into intense tissue browning and deterioration which is
reflected in reduced capacity for protein synthesis and changes in the free amino acid pools
and protein patterns. Oxidized products, such as quinones, are known to be highly reactive
and inhibit enzyme activity leading to the death of the explants. The toxicity of the
oxidized compounds to larvae feeding on a brown callus caused death or reduced growth.
As a consequence of browning, tissue senescence, recalcitrance in embryogenesis and
regeneration have been observed [61].
2.3.3 The Difficulty in Using Explants from Mature Trees
There are numerous substantive phenotypic traits associated with juvenility, but they vary
considerably among species. Commonly, the leaves on young plants are of a different
shape than those on mature parts and may be simple rather than compound (or occasionally
the reverse); juvenile leaves may also have a special type of cuticle and be arranged with a
distinct phyllotaxy. Compared to their adult counterparts, young plants may have a
modified resistance to pests and diseases. Juvenility in woody plants is often manifested by
prolonged vigorous shoot growth [75].
Young healthy tissues that are rich in nutrients, and possibly endogenous hormone, are the
best choices for the induction of cell division. While the woody plant material is generally
a poor choice. Also, plant tissues that are high in oxidase activity pose a special problem
since enzymatic browning retards cell division. The browning results from the activity of
wound-induced copper oxidases (polyphenoloxidase). This may be suppressed, to some
extent, by the use of an antioxidant mixture [15].
Juvenile plants have a different growth habit, leaf shape, and an enhanced ability to form
adventitious roots. When petioles from the juvenile form were excised and treated with
auxin in vitro, cortical parenchyma cells adjacent to the vascular bundles divided and
formed root primordia. However, when petioles from adult leaves were treated in a similar
manner, callus formed and some callus cells divided to form root primordia. The juvenile
form had pre-existing competent cells that were able to respond to auxin and become
determined to form roots. However, the adult form appeared to lack cells with pre-existing
competence to form roots, but competence was acquired by some callus cells once they
had been initiated [75].
Explants taken from mature shoots are frequently more liable than juvenile material to
suffer necrosis, especially when surface disinfested and placed in culture, and juvenile
explants are usually more readily established in vitro and grow and proliferate at a more
rapid rate than adult material. This is particularly true with tree species where
micropropagation of adult material is often difficult. Juvenile plants frequently grow more
rapidly and have stronger apical dominance than adult forms. However, shoot explants
from juvenile plants generally proliferate more axillary shoots than shoot explants from
adult forms [19].
A safer and more promising alternative would be to use other plant parts such as apical
buds and apical meristems for establishment of cultures. The apical bud is defined as the
bud located immediately below the apical meristem and enclosed within the leaf sheath.
Selecting these young tissues makes it possible to reduce infection since the apical zone
displays better aseptic conditions because of the reduced size of the explant and the small
area exposed to the external environment [57].
2.3.4 High – Temperature Degradation of Media Components
Several chemicals employed in plant tissue culture media degrade on exposure to steam
sterilization. Gibberellins are rapidly degraded by high temperatures, and biological
activity of a freshly prepared solution of GA3 was reduced by more than 90% as a result of
autoclaving. The auxins IAA, á-NAA, and 2,4-D are relatively thermostable depending on
the inorganic basal medium and supplements. Aqueous solutions of kinetin, zeatin, and 2iP
have been chromatographed on thin-layer silica-gel chromatograms before and after
prolonged autoclaving with no breakdown products detected [15].
Vitamins have varying degree of thermolability. Most workers autoclave the vitamins with
the remainder of the medium. Nicotinic acid, pyridoxine, and thiamine in a MS liquid
medium (PH 5.5-5.6) showed no signs of degradation after autoclaving; nevertheless,
thiamine is rapidly destroyed if the PH of the medium is much above 5.5. Calcium
pantothenate can not be autoclaved without destruction. If the research study involves
vitamin activity, then the vitamins should be sterilized by microfiltration. Since those
employed in most our experiments are apparently thermostable, the vitamin supplement
will be added to the medium prior to autoclaving [15].
One of the most frequently employed carbohydrates in media is sucrose. This disaccharide
decomposes, to some extent, on autoclaving to release a mixture of D-glucose and Dfructose. A recent study indicated that 5 percent of the sucrose in an MS liquid medium
was hydrolyzed during autoclaving. This degradation can be inhibitory to some cultured
tissues. Presumably the toxicity is due to the degradation products of D-fructose. Steam
sterilization may also catalyze reaction within the media between carbohydrates and amino
acids [15].
Among various alternatives for reducing costs in so-called ‘‘biofactories’’, or commercial
plant production laboratories, a potentially good alternative is to replace the autoclaving
procedure described by some researchers such as using a microwave oven, and use of
sodium hypochlorite or hydrogen peroxide with a low concentration in the media [76].
2.3.5 Hyperhydricity
Vitrification was first used as a term in conventional plant tissue culture to describe a
morphological response of plant tissues to stresses [77]. The symptoms were water soaked,
thick, elongated, wrinkled, curled, brittle and translucent leaves, shoots with shorter
internodes, and the low number of shoots/explant. However, as a term vitrification is
confusing, because in cryobiology vitrification (cryopreservation) describes the process
where plant tissue is converted into ice during freezing preservation. Thus, later the same
author suggested that the term vitrified should be substituted for hyperhydric [61].
Anatomically hyperhydric leaves have large vacuolated mesophyll cells showing a
hypertrophy of the cells with large intercellular spaces filled with water instead of air.
Tissues have less lignin and the vascular system is abnormal. Leaves have less if any
surface wax or the wax has an altered chrystalline structure, and thin cuticle with a lower
level of cutin, pectin and cellulose. Several abnormalities have been observed also in
hyperhydric cells. The chlorophyll content has been shown to be low and to contain
abundant plastoglobuli. The guard cells contained high levels of K+ and were
morphologically abnormal as well as the stomata them self. Also the photosynthetic rate
was lower in hyperhydric cells compared to normal tissue [61].
Biochemically the activity of several enzymes is altered in hyperhydric leaves. The activity
of superoxide dismutase was high in hyperhydric shoots resulting in hydrogen peroxide
(H202) accumulation. Also, the blockage of the porphyrin pathway and lack of H202
detoxifying enzymes (catalase, ascorbate peroxidase, and glutathione reductase). And a
deviation of the nitrogen metabolism induced by NH3 towards polyamines caused the
formation of toxic oxygen forms [77, 78].
It is unlikely that nitrate level alone affects hyperhydricity, although plantlets showed
changes in protein synthesis, which correlated both positively and negatively with nitrate
content [61]. It was also observed that habituated (capable to induce callus without
exogenous growth substances) and hyperhydric calli resembled each other biochemically
[78].
Development of the morphological symptoms of hyperhydricity depends on many factors.
Hyperhydricity has been observed to increase by zeatin, fructose, thiadiazuron, low sucrose
concentration, BAP, and high relative humidity in the culture vessel. Visual symptoms
occur only for a certain period of time and may vary between species. The visual
symptoms can be reduced by controlling various factors. Medium components or growth
conditions such as silver nitrate, yeast extract, potato juice, increased gelling agent, ABA,
high KNO3 : NH4Cl2 ratio, phloridzin , paclobutrazol, increasing gas exchange in the
culture vessel, and cold or reduced temperature treatments and increased Ca concentration
have decreased the abnormal hyperhydricity of tissues [61].
2.3.6 Shoot Tip Necrosis
Calcium deficiency in plants results in poor root growth and in the blackening and curling
of the margins of apical leaves, often followed by a cessation of growth and death of the
shoot tip. The latter symptoms are similar to aluminum toxicity. Tip necrosis has been
especially observed in shoot cultures, sometimes associated with hyperhydricity. It often
occurs after several subcultures. After death of the tip, shoots often produce lateral
branches, and in extreme cases the tips of these will also die and branch again [19].
Different approaches have been reported to overcome the loss of cultures by different
means including more frequent subculture, using modified media by addition of silver
nitrate, GA3, fructose and calcium [79].
Calcium, as a component of cell walls, membranes and lignin, protects membranes from
damage and leakiness, thus maintaining cell integrity and membrane permeability.
Calcification strengthens plant cell walls and enables increased plant resistance to
pathogen infection. Mitigation of calcium deficiency during shoot multiplication in potato
was achieved by the use of calcium-rich media, and by periodically inverting of the culture
tubes containing liquid medium supplemented with calcium gluconate for better interaction
of the explant with the medium [79].
As calcium is not remobilized (nontranslocatable) within plant tissues, actively growing
shoots need a constant fresh supply of ions in the transpiration stream. An inadequate
supply of calcium can result from limited uptake of the ion, and inadequate transport, the
latter being caused by the absence of transpiration due to the high humidity in the culture
vessel (George et al, 2008). And can introduce undesirable anions. A temporary increase
in Ca2+ concentration to 1 or 10 M does not significantly alter the ionic environment
within the cell, but is yet sufficient to trigger fundamental cell process such as polarized
growth, response to gravity, plant growth substances, cytoplasmic streaming, and mitosis
[79].
Addition of higher levels of calcium chloride in shoot multiplication as well as rooting
medium did not result in complete alleviation of necrosis and this may be due to chloride
toxicity caused by the supra-optimal level of calcium chloride in the medium. Excess
calcium may also produce deficiencies in magnesium and potassium. Although calcium
can be present in millimolar concentrations within the plant as a whole, calcium ions are
pumped out of the cytoplasm of cells to maintain the concentration at around only 0.1 M.
This active removal of Ca2+ from the protoplasm is necessary to prevent the precipitation
of phosphate and interference with the function of Mg2+ [2].
A remedy can sometimes be obtained by reducing the culture temperature so that the rate
of shoot growth matches calcium supply, using vessels which promote better gas exchange
(thereby increasing the transpiration and xylem transport), or by increasing the
concentration of calcium in the medium. The last two remedies can have drawbacks: the
medium will dry out if there is too free gas exchange [19].
2.3.7 Light Requirements
The illumination of plant cultures must be considered in terms of intensity, length of the
daily exposure period, and quality. At the outset, it should be clearly noted that the light
requirements of tissue cultures are not the same as those of autotrophically developing
whole plants. In tissue culture, photosynthesisis not a necessary activity, except perhaps
during the latter part of Stage III, since carbohydrate is adequately provided. Nevertheless,
light is needed to regulate certain morphogenetic processes. It has been reported to be
important for the formation of shoots, the initiation of roots, the differentiation of
cladophylls, and in asexual embryogenesis. Instances of failure of tissue cultures are
sometimes caused by the use of plant growth chambers of similar facilities where the light
provisions have been intended for autotrophic plant development. The difficulty most often
encountered with such facilities is the excessively high light intensity; sometimes the
quality of illumination is also unsuited for tissue culture [10].
2.3.8 The Cost of Generating Plantlets
The cost of generating plantlets is very high. The most advanced micropropagation
technology is still labour and cost intensive. Therefore, this process can only be profitable
if species cannot be multiplied conventionally and a high return is expected from genetic
gain, yield, disease-free stock, etc [11].
2.3.9 Somaclonal Variation
In addition to the variants/mutants (cell lines and plants) obtained as a result of the
application of a selective agent in the presence or absence of a mutagen, many variants
have been obtained through the tissue-culture cycle itself. These somaclonal variants,
which are dependent on the natural variation in a population of cells, may be genetic or
epigenetic, and are usually observed in the regenerated plantlets. Somaclonal variation
itself does not appear to be a simple phenomenon, and may reflect pre-existing cellular
genetic differences or tissue culture- induced variability. The variation may be generated
through several types of nuclear chromosomal re-arrangements and losses, gene
amplification
or
de-amplification:
non-reciprocal
mitotic
recombination
events,
transposable element activation, apparent point mutations, or re-activation of silent genes
in multigene families, as well as alterations in maternally inherited characteristics [80].
Many of the changes observed in plants regenerated in vitro have potential agricultural and
horticultural significance. These include alterations in plant pigmentation, seed yield, plant
vigour and size, leaf and flower morphology, essential oils, fruit solids, and disease
tolerance or resistance. The same types of variation obtained from somatic cells and
protoplasts can also be obtained from gametic tissue [80].
One of the major potential benefits of somaclonal variation is the creation of additional
genetic variability in coadapted, agronomically useful cultivars, without the need to resort
to hybridization. This method could be valuable if selection is possible in vitro or if rapid
plant-screening methods are available. It is believed that somaclonal variants can be
enhanced for some characters during culture in vitro, including resistance to disease
pathotoxins and herbicides and tolerance to environmental or chemical stress. However, at
present few cultivars of any agronomically important crop have been produced through the
exploitation of somaclonal variation [80].
Although somaclonal variation can be used as a source for variation to obtain superior
clones, it can be also a very serious problem in the plant tissue culture industry resulting in
the production of undersirable plant off – types [80].
2.3.10 Acclimatization of Tissue-Cultured Plants
A substantial number of micropropagated plants do not survive transfer from in vitro
conditions to greenhouse or field environment. The greenhouse and field have substantially
lower relative humidity, higher light level and septic environment that are stressful to
micropropagated plants compared to in vitro conditions. The benefit of any
micropropagation system can, however, only be fully realized by the successful transfer of
plantlets from tissue-culture vessels to the ambient conditions found ex vitro. Most species
grown in vitro require an acclimatization process in order to ensure that sufficient number
of plants survive and grow vigorously when transferred to soil [81].
The transfer of in vitro plantlets to ex vitro conditions is one of the most critical factors of
the micropropagation process and a cause of higher production costs.
High mortality is often observed upon transfer to ex vitro conditions as the cultured plants
have a poorly developed cuticle, nonfunctional stomata and a weak root system (Table 3).
In order to increase growth rate and reduce mortality of plantlets at the stage of
acclimatization, recent research has focused on control of the environmental conditions.
One approach has been to modify the environmental conditions during acclimatization by
increasing light intensity or both increasing the light levels and altering the CO2
concentration. Another approach has been to change the environment during the
multiplication and rooting stages, including increasing light intensity and CO2
concentration in culture tubes, and decreasing the sugar concentration. All were found to
be beneficial for plantlet growth in the later stages of micropropagation [82].
A biological approach to reducing the stress of acclimatization and providing faster growth
of micropropagated plantlets is the establishment of vesicular-arbuscular mycorrhizae
(VAM) on micropropagated plantlets during acclimatization. VAM colonization of
horticultural plant roots can improve growth by increased uptake of phosphorus, zinc and
other minerals and may reduce the incidence of disease. Moreover, colonization with VAM
fungi may increase transplant uniformity and reduce both transplant mortality and injury.
Recent work has also shown improvement in water relations of the host plant using VAM.
These fungi may also improve drought tolerance by decreasing leaf water potential, by
reducing stomatal and root hydraulic resistances, and by increasing transpiration rates.
Several studies have focused on VA mycorizal formation during acclimatization of cultural
plantlets [82].
Table 3: Anatomical and histological differences between roots, stems and leaves of
plants in vitro and after transplanting ex vitro. George et al. [19].
Plant Organ and Tissue
Roots
Epidermis
Cortex
Root hairs
Stems
Epidermis
Cortex
Leaves
Epidermis
Cuticle
Stomata
Spongy Parenchyma
Chloroplasts
In vitro
Ex vitro
Thin
Uniseriate
Broad,
irregular
enlarged,
hypertrophied
individual
cells,
numerous intercellular spaces; loose
arrangement of cortical arenchyma.
Few or no, thick, short and straight,
fine, delicate appearance, many
fused together, usually abundant.
Small diameter.
Limited development.
Limited
development,
little
collenchyma, few sclerenchyma
fibers; starch grains in old stem.
Small, succulent, brittle and
hyperhydrated.
Deformed thin cell walls irregularly
shaped.
Thin and discontinuous.
Large root system
Uniseriate and multiseriate
Irregular guard cells with thin cell
walls, large stoma.
Highly vacuolated with large
intercellular air-spaces.
Low chlorophyll, abnormal nonfunctional chloroplasts, limited
formation of grana, abundant
stroma, and starch grains.
Uniform, compact cortical
cell arrangement
Long, thin, slender, fibrous,
wiry and formed an
interwoven mat.
Large diameter.
Fully developed.
Fully
developed
with
continuous cylinder of
collenchyma.
Normal shape and size
Normal cell walls.
Thick and continuous.
Normal guard cells.
Normal cells with regular
air-spaces.
Normal chloroplasts with
typical granal structure,
increased
chlorophyll
content.
2.4 Use of Charcoal in in vitro Culture
Effects of charcoal on tissue response in in vitro culture appear to be dependent not only on
the kind of charcoal and their degree of activation, but also on the plant species cultured.
The addition of activated charcoal to tissue culture media may have either a beneficial or
an adverse effect on growth and development, depending upon the medium, tissue used,
and/or objective(s) of the researcher [83].
Use of charcoal can make a major difference in the success or failure of a given culture
attempt. In general, effects of charcoal on in vitro cultures could be dealt with under the
following headings:
2.4.1 Providing a dark Environment
Charcoal provides a degree of darkness during in vitro culture. Light is a major factor of
the culture environment and has been shown to have an effect on organized development in
vitro. Light requirements for differentiation involve a combination of several components,
including intensity, daily light period and quality. The properties of light affect tissue
cultures, and influences their growth and development. Reduction of light at the base of a
shoot can provide an environment conducive to the accumulation of photosensitive auxin
or co-factors.
Addition of activated charcoal in the rooting medium improved the potential for
adventitious rooting, not only in terms of rooting rates, but also an enhancement of the
number and the length of the roots, as well as root score. This stimulating effect of
activated charcoal on root ability was particularly associated with mature explants.
Charcoal may affect the activity and/or stability of plant growth regulators by reducing or
excluding light in in vitro culture. Light promoted degradation of IAA and IBA in both
liquid and solid agar media [83].
2.4.2 Adsorption of Undesirable or Inhibitory Substances in in vitro Cultures
It is widely accepted that some of the beneficial effects of activated charcoal can be
attributed to the removal of inhibitory substances from the media, produced either on
autoclaving the media [84] or released by the tissue itself [85]. Charcoal may be able to
adsorb toxic brown/black pigments (phenol-like compounds and melanin) as well as other
unknown colourless toxic compounds. Fridborg et al. [85] found that differentiation
occurred in Daucus and Allium cultures in activated charcoal-containing media. The media
without activated charcoal contained phenolic compounds and other metabolites which
inhibited embryogenesis and morphogenesis.
A major breakdown product of autoclaved sugars, which has been shown to reduce growth
of plant tissue in vitro, is HMF (5-hydroxymethyl-2-furaldehyde).
The inhibition of growth of tobacco anther cultures by 100 mg \ L HMF was eliminated by
addition of activated charcoal, while the addition of 0.3% activated charcoal increased
plantlet yield by 200–300% in all the anther cultures [84].
It is normal for the pH of the medium to drift. Typically, during tissue culture experiments,
pH is adjusted prior to autoclaving to between 5.2 and 5.8. Tissue culture media are poorly
buffered and in the presence of developing tissue the pH may go up or down. Johansson et
al. [86] showed that the pH of the medium had increased by almost 0.5 pH units in one
hour. After eight hours the pH appeared to be reasonably stable. As stated previously,
however, AC may have acidic character depending on how it was manufactured.
2.4.3 Adsorption of Plant Growth Regulators in In Vitro Culture
The concentration and combination of auxins and cytokinins in the nutrient medium is
usually a key factor which determines successful plant regeneration. The use of activated
charcoal as a culture component for adsorption of toxic plant metabolites is known.
Activated charcoal is able to adsorb high concentrations of the growth regulators BA, IAA,
IBA, NAA and Kinetin, in both liquid and solid media [87].
Ethylene is a gaseous hormone produced by plant tissue and/or the medium. Activated
charcoal was able to adsorb ethylene released by the medium and by Nicotiana tabacum
nodes [88]. More ethylene was produced by Anemone caronaria seedlings than agar alone
[89]. The authors also noted that the effect of activated charcoal on ethylene is dependent
on culture conditions, such as container volume and shape, medium volume, and surface
exposed to the inner atmosphere.
Takayarna et al. [90] reported that root formation and growth were inhibited by higher
concentration of BA, but this inhibition was completely reversed by the addition of
activated charcoal.
Root formation and growth were better in BA-free medium containing activated charcoal
than in the medium without activated charcoal. Similar results were observed in bulb
formation. This could be due to activated charcoal regulating internal physiological
processes [83].
2.5 Strawberry Tissue Culture
One of the important goals of the agricultural policy in the world is to increase the acreage
of strawberry to meet the demand of local fresh market, processing and export. Importing
mother plants is expensive. Healthy stocks used for propagation through conventional
methods are not available.
Micropropagation of strawberry plants were introduced in 1974 (Boxus) [91].
Immediately, the most important European nurseries producing several millions plants per
year, were interested in this technique. The two basic reasons that gave the importance to
this technique were: it gave a definitive answer to the problems of soil fungi, causing a lot
of damage to the strawberry fields and by another way, tissue culture plants seemed to
produce more runners per mother plant in a short time. However, strawberry tree is
difficult to propagate by seed due to genetic variation and specific requirements of seed
germination. In addition, rooting percentage of cuttings is relatively low [92].
In general, strawberry can be managed easily under in vitro conditions. Meristem tips,
generally obtained from runners of virus-free plants, are commonly used to establish in
vitro cultures, which are employed for mass propagation or as a source of plant material for
regeneration and transformation experiments [43].
Micropropagation of strawberry by axillary buds has been studied intensively for a long
time [93]. On a commercial scale, tissue culture-derived strawberry plants are estimated to
cost four to five times more than plants produced by conventional propagation [6].
However, micropropagated strawberry has several advantages, such as its ability to
multiply virus-free stock rapidly and, in particular, the improved capacity of these plants to
produce runners for planting in the field [94, 6]. On contrast, when used directly in the
field for fruit production, micropropagated material shows a decrease in fruit size and
weight [94].
Several parameters used during the in vitro phase can affect the behavior of
micropropagated strawberry in the nursery, e.g. plant genotype, mineral formulation, type
and concentration of cytokinin in the medium and the number of subcultures. It is
generally recommended not to exceed four to five subcultures to avoid loss of trueness-totype of the propagated material [54].
This technique is useful in case of the introduction of new cultivars. Moreover, the storage
of tissue cultured propagules requires less space than traditional runner plant and the in
vitro storage can be initiated at any time during the production cycle [95].
Prior experiences with strawberry micropropagation indicate that vitro plants are more
uniform, produce higher number of runners, have better survival in the field, and the fruit
yield increases in 24% than plants propagated by the traditional method[96].
Another advantage of micro-propagation is the elimination of pest and pathogen stress
during the production cycle, assuming that the initial stock plant is free of diseases.
Therefore tissue culture technique was applied to evaluate its feasibility for a wider use.
Several authors have dealt with this subject from several aspects. Boxus 1983, [97]
reported that each m2 of growing area of strawberry can produce 40000 plantlets year-1,
but Abramenko 1983, [98] has cultured apical meristem of strawberry and found that from
one meristem 3000 shoot primordial were produced in 6 months and half of these
developed into normal plants. These plants were vigorous and after transplanting in the soil
some produced up to 500 new runner plants and found that new runner plants were 10
times more than that produced by conventional material.
The production program suggested by Vit et al. [99] for commercial planting material is
based on the selection of economically important clones; freeing these of virus by a
combination of heat therapy and meristem culture. Tissue culture aimed particularly at the
rapid propagation of breeding material [100].
2.5.1 Strawberry Organogenesis
2.5.1.1 Strawberry Direct Organogenesis
Most commonly used explant for strawberry micropropagation is the meristem from the tip
of runners. The explant is placed on a medium containing no or low levels of auxins and
higher levels of cytokinins to promote axillary budding while preventing callus formation.
The cytokinins are used to overcome apical dominance and enhance the branching of
lateral buds from the leaf axis. Additional shoots are produced through further axillary bud
growth [101].
Strawberry Fragaria × ananassa Duch. Was the first micropropagated in vitro on a
commercial scale by Buxus in 1974. There after this technique has been widely used
especially in Europe and USA.
In 1974, [91] Boxus experimented with BA to determine its influence on shoot and root
production in vitro. He established that shoots would proliferate in the presence of the
cytokinin but roots would not form until the explant was without cytokinin. He concluded
with the statement that micro-propagated strawberry plants would replace traditional
methods of propagation for the commercial trade.
Mullin et al. [102] grew strawberries with strawberry mild yellow edge (SMYE) viruses
for 6 weeks in a 36°C growth chamber before excising 0.3 to 0.8 mm meristematic tips
with leaf primordia. The result was that 33% to 75% of the resulting plants were SMYEfree according to leaf insert graft indexing to indicator strawberry plants.
Liu and Sanford [103] reported that the methods were developed for efficient shoot
regeneration from leaf and runner tissues of the strawberry Fragaria x ananassa Duch.
cultivars ‘Allstar’ and ‘Heneoye’. They showed that optimal regeneration condition
differed for ‘Allstar’ depending on whether leaf tissue was derived from plantlets grown in
vitro or from plants grown in the greenhouse. ‘Allstar’ leaf tissues derived from in vitro
culture regenerated shoots most efficiently in a Linsmaler- Skoog (LS) medium containing
2.5 mg BA and 0.5 mg IBA /liter, ‘Allstar’ leaf tissues derived from greenhouse plants
regenerated shoots best in (LS) medium containing 3.0 mg BA and 0.1 mg IBA /Liter.
Addition of casein hydrolysate (CH) at either 400 or 600 mg/l stimulated shoot production.
A supplement of KNO3 at 2000 mg/l also enhanced regeneration efficiency of greenhouse grown leaf tissues. ’Hsotoye’ had lower regeneration potential in most treatments than
‘Allstar’ and only produced shoots in a (LS) medium containing 5.0 mg BA, 0.5 mg IBA,
and 400mg CH /Liter. Shoots from runner tissues of both cultivars were best obtained
using (LS) medium containing 10.0 mg BA, 2.0 mg IAA, and 500mg CH /Liter. Shoots
proliferation from runners was dependent on the diameter of the runner, with diameters of
more than 2.0 mm having poor regeneration potential.
Boxus 1989, [104] claimed that this clonal propagation process proceeded exclusively
through axillary branching which normally leads to true-to-type progenies.
Jelenkovic et al. [105] studying different cultivars than Nehra or Finstad studied, tested
hypocotyls, runners, petioles and lamina. Only young, fully expanded leaves were used in
the lamina study. They determined in preliminary tests that BA and 2, 4-D were the most
effective phytohormones to use. Regeneration was noticeably lower in inoculated explants
than in controls possibly due to the explants dying from the toxic effects of the bacteria.
The researchers expressed frustration over the inconsistency of response of the same
explant material exposed to the same conditions.
Lopez - Aranda et al. [94] they studied in vitro culture of strawberry cvs. ‘Douglas’ and
‘Chandler’ for effects of mineral salts, BA levels and the number of times an explant was
subcultured on the regeneration capacity. The field behavior of the regenerants was also
tested in the comprehensive investigation.
The best quality regeneration was achieved with a formulation of N30K salts formulation
for both cultivars. Best results on rooting were obtained with either the Knop or N30K salts
formulation. Addition of 500 mg/l activated charcoal also enhanced rooting; in contrast,
high BA levels during multiplication decreased the rooting capacity of shoots. They noted
that micropropagated plants showed a higher runner production capacity than their control
counterparts. This increase in quantity was not linked with decreased quality. They also
concluded that salts formulation, BA levels (1.48 µM - 4,44 µM) and the number of the
subcultures (1-8) seemed not to affect the behaviour of plants in the nursery. No adverse
effects on total fruit production or fruit size could be associated with the mineral
formulation used during the in vitro phase. High BA concentrations adversely affected
total fruit production although differences between treatments were not significant.
The authors concluded that the field results of micropropagated plants are greatly
influenced by their in vitro culture.
Jemmali 1994a, [106] suggested that Stipular buds (SB) formation occurred spontaneously
on Gorella strawberry leaf stipules during proliferation phase on Boxus medium. These
buds gave rise to normal shoots on the inner median zone between the stipule tips. Some
stipular buds also have been observed on other parts of adaxial surface of the stipule.
Stipular bud formation took place directly on the stipule without an intermediate callus.
The first stage consisted of subspherical pretruions of small cells which progressively
differentiated into shoots that were able to prolifrate and to root. Scanning microscopy was
used to examine the developmental stages of this adventitious organogenesis and to
describe morphologic abnormalities, e.g, multiapex formation and fasciation. In vitro
cloning of plantlets was made from axillary and SBs produced by the same tufts. The
greater flowering abundance of stipular plants resulted in a drastic reduction of the
commercial production and the fruit caliber when compared to axillary plants. However,
the general phenotype of the mother cultivar (Gorella) was not affected in either case.
Nehra et al. [107] They studied the Effect of in vitro propagation methods on field
performance of two strawberry cultivars, ‘Redcoat’ and ‘Veestar’, propagated by meristem
culture (MC), callus culture (CC) and direct shoot regeneration (DR) from leaf disks were
compared for their vegetative and reproductive characters with standard runner (SR)
propagated plants under field conditions. They observed that in the planting year, in vitro
propagated plants of both cultivars had the same number of leaves as SR plants, but in
vitro propagated ‘Redcoat’ produced fewer stolons per plant than SR plants. However, in
the following year, in vitro propagated mother plants of both cultivars had more leaves and
higher runner production than SR mother plants. Flowering and fruiting behavior of
‘Veestar’ was not appreciably influenced by in vitro propagation methods. However, in
vitro propagated plants of ‘Redcoat’ flowered earlier and produced more flowers and fruits
than SR plants, but still maintained normal berry weight. Among in vitro propagated
plants, DR plants of ‘Redcoat’ were the earliest to flower, whereas MC plants produced
more flowers and fruits. The field performance of the first daughter plants derived from the
in vitro propagated plants was consistent with their respective mother plants. They also
observed that leaf shape of both cultivars was not altered by in vitro propagation.
Phenotypic abnormalities were mainly confined to occurrence of yellow leaf variants in
MC and CC plants and occasional appearance of plants with irregular flowering and
growth habit among CC plants.
Jemmali et al, (2002)[37] demonstrated
that
adventitious
stipular
bud formation
occurred in vitro in many strawberry cultivars during the proliferation phase on medium
containing Knop macronutrients, MS
micronutrients, vitamins, amino acids, 2.22µM
BAP, 2.46µM IBA and 0.29 µM GA3. As described previously for cultivar Gorella,
cultivar Elsanta also showed adventitious stipular buds developing on the abaxial median
zone between the stipule tips. To compare the shoots produced from both types of buds,
clonal propagation was initiated from stipular buds and from axillary buds on the Knop
macronutrients and MS micronutrients medium. Stipular buds were separated from the
meristem-tip initiated plantlet and cultivated in the presence of a lower BAP concentration
(1.33 µM) to prevent further stipular bud formation. During proliferation cycles, stipular
originated propagules were very easily distinguished by their specific leaf phenotype and
light green colour in comparison to plantlets cloned for an axillary bud. Their
multiplication rate and cytokinine content were also higher than for axillary buds. No
significant diference was observed in auxin content.
Meria et al. [108] demonstrated that actively growing shoots of potted greenhouse - grown
strawberry tree( Arbutus unedo L.) were initially sterilized and established in basal woody
plant medium containing 11.1 µM BA. Optimum shoot proliferation was achieved on a
basal WPM containing MS vitamins, sucrose, agar and 22.2 µM BA. They observed in
preliminary testes with MS basal salts instead of WPM basal salts resulted in extensive
oxidative browning and failure of explants after culturing for 3 weeks, This browning was
due to a high concentration of salts or, in particular, due to high NH4NO3 concentration in
MS medium. Microshoots rooted successfully in basal in vitro medium containing 10 µM
IBA or IAA, but their survival rate during acclimatization was low. Addition of a mixture
1 part peat: 4 parts perlite in the basal in vitro rooting medium (1:1 v/v) containing 10 µM
IAA resulted in high rooting percentage and plantlets with branched roots. These plantlets
were successfully acclimatized. This novel rooting medium can be exploited further due to
its potential in commercial applications.
Zhou et al. [109] found that rooting strawberry plantlets under photoautotrophy conditions
significantly increased plant height and weight after acclimatization. This effect was
related to a better photosynthetic performance of in vitro plants.
Biswas et al, [110] studied about the effects of colour illumination on multiple shoot
regeneration from runner tip explants of strawberry. Six colour (mixed, white, red, yellow,
blue and green) illuminations were used in this study. They observed that the mixed colour
illumination showed the high percentage of shoot proliferation. Fresh and dry weights were
also significantly higher under the mixed colour condition. Proliferated shoots showed
100% rooting in half strength of MS media. Plantlets were established successfully in soil.
Madany et al. [111] suggested that an efficient method of micropropagation based on an
increased percentage survival of explants and reduced phenol-induced browning in
strawberry (Fragaria x ananassa Duch.) cv. Camorosa and Selva has been developed. The
effect of hormone concentration growth regulator balance and kind of antioxidant in
medium on the direct shooting of meristem culture was studied. They showed that the best
results were obtained on MS basal medium with B-5 vitamins supplemented with 1 mg/l
IAA and 2 mg/l BAP for Camorosa and 2 mg/l IAA and 2 mg/l BAP for Selva. Use of 0.2
% activated charcoal was better than 2% PVPP to eliminate inhibitory substances from in
vitro cultures. Excised shoots rooted on MS medium with 2 mg/l IAA and 0.3 mg/l BAP.
Sakila et al. [112] showed that nodal segments of strawberry gave rise to multiple shoots
when cultured on MS medium supplemented with different concentration of BA with KIN
or GA3. The highest response of shoot multiplication was obtained in MS containing 1.5
mg/l BA + 0.5-0. mg/l KIN. The regenerated shootlets were rooted on MS basal medium
with different concentrations IBA and IAA. The maximum frequency of rooting and
highest number of roots was produced on medium containing 1.0 mg/l IBA. The plantlets,
thus developed were hardened and successfully established in soil. The plants raised
through tissue culture exhibited normal growth, flowering and fruit setting.
Emarah 2008, [36] demonstrated that an efficient method for shoot regeneration, root
formation from runner tips and acclimatization of strawberry plantlets was developed.
Runner tips of 1-2 cm long were used as source of explnts. After surface sterilization apical
meristems of 3-5mm long were isolated and used as explnts. At multiplication stage,
results indicated that the highest vegetative parameters (shoot number, shoot length and
leaf number) were observed when MS medium supplemented with 1 mg/l BA followed by
the medium contained 1 mg/l BA and 0.1 mg/l IBA. However control treatment showed a
significant similar result in shoot length only. Results of this study indicated that, BA was
more effective in enhancement the growth of strawberry in vitro compared to Kin and
thidiazuron. He also demonstrated that at rooting stage, it was clear that MS medium at full
strength containing 30 g/l sucrose significantly surpassed all other combinations of MS
strengths and sucrose concentrations in increasing root number and length per plantlet and
fresh weight/plantlet. The same treatment enhanced the shoot length but without significant
difference compared to some other combinations. The treatment contained 3 g/l agar with 6
g/l perlite significantly enhanced root formation (number and length) as well as shoot
length, fresh weight/plantlet, and leaf number/plantlets. Finally, plantlets were successfully
acclimatized and the soil mixture contained peatmoss: perlite (2: 1, V/V) observed high
percentage of survival of plants (80%) with enhancing both root number and
length/plantlet, plantlet height and leaf number/plantlets.
2.5.1.2 Adventitious Shoot Regeneration
Adventitious shoot regeneration has been achieved in several cultivars using a broad range
of explants. These include leaf explants, petioles, stipules, stem tissue, runners, mesophyll
protoplasts, anthers, cotyledons, roots and immature embryos [43].
Jemmali et al. [113] focused that adventitious buds of strawberry ‘Gorella’ arise between
the two leaf stipule points and having different numbers of subculture when these
adventitious shoots are into contact with Boxus medium, they proliferate fastly by axillary
branching. They concluded that the performances of the plants regenerated from stipular
shoots are similar to the control ones concerning DNA ratio and preliminary analysis by
flow cytometry gave no modification of their ploidy level, epigenetic or genetic variations
are not excluded, the plants used as control are indeed a mixture of different shoot origin
and that can explain why they did not observe real difference between the treatments. And
they noted also that all roots of stipular plantlets were exclusively white, whereas the
control ones were more or less colored with anthocyanin. This observation was confirmed
in another experiment and could be used as a marker if it remains stable under variable
culture condition.
Rugini and Orlando [114] studies about adventitious shoot regeneration by used stipules
explants, they found great differences in shoot regeneration ability from calluses among
cultivars when leaf, petiole and root tissues were used as initial explants. However, these
differences disappeared when whole leaves, including stipules, were used as explants.
Passey et al. [115] studied seven commercial cultivars of strawberry using leaf disks,
petioles, roots, and stipules as explant material. They started out by establishing and
growing runner tips in vitro and subculturing them every three weeks until enough material
was produced to begin the experiments. The leaf disks had the highest regeneration rates
for all cultivars with greater than 90% of explants producing shoots.
Samir 2005, [116] reported an efficient system to regenerate shoots on excised sepals
(calyx) of greenhouse-grown ‘Bounty’ strawberry (Fragaria - ananassa Duch.) was
developed in vitro. He show that Sepal cultures produced multiple buds and shoots without
an intermediary callus phase on 2–4mM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea
(thidiazuron, TDZ)-containing shoot induction medium within 4–5 wk of culture initiation.
And he also shows that young expanding sepals with the adaxial side touching the culture
medium and maintained for 14 days in darkness produced the best results. In a second
experiment, he observed that sepals proved more effective than the leaf discs and petiole
segments for regenerating shoots. A third experiment he compared the effects of six
concentrations of two cytokinins (TDZ at 0, 0.5, 2, and 4mM and zeatin at 2 and 4 mM) for
elongation of sepal-derived adventitious shoots. The results indicated that the media
containing TDZ generally promoted more callus formation and suppressed shoot
elongation. TDZ-initiated cultures transferred into the medium containing 2–4mM zeatin,
produced usable shoots after one additional subculture. And shoots were rooted in vitro in
the same medium used for shoot regeneration, but without any growth regulators. When
transferred to potting medium,
85–90% of in vitro plantlets survived.
Qin et al. [117] reported that ‘Toyonoka’ strawberry leaf explants cultured for 10 days in
shoot regeneration medium in the presence of AgNO3 not only enhanced shoot
regeneration efficiency but also expedited the inhibition of adventitious buds. Being an
ethylene inhibitor, AgNO3 can markedly promote organogenesis in strawberries.
Samir 2006, [118] worked about the effects of 0, 2, 4 or 8 ìM thidiazuron (TDZ) and
explant type on adventitious shoots regenerated on excised leaf disks, sepals (calyx) and
petiole halves of greenhouse-grown ‘Bounty’ strawberry (Fragaria - ananassa Duch.). He
saw that a moderate concentration (2 – 4 ìM TDZ) supported bud and shoot regeneration
without an intermediary callus phase on young expanding sepals, or on leaf disks, or on
petiole, all maintained for 14 d in the dark. Sepals proved more effective than leaf discs
and petiole segments for regenerating shoots. Another experiment, he was compared the
effects of 0, 0.5, 1, 2 and 4 ìM TDZ or zeatin on shoot proliferation and rooting of sepalderived adventitious shoots. The results indicated that the media containing TDZ generally
promoted more callus formation and suppressed elongation and rooting of shoots. And
Shoots proliferated and roots developed best when explants were cultured in medium
supplemented with 1 or 2 ìM zeatin.
2.5.2 Strawberry Indirect Organogenesis
Kartha et al. [119] successfully regenerated ‘Redcoat’ using a combination of BA, IBA and
GA3 as a precursor to a cryopreservation study of the cultivar. Kartha et al. (1980) studied
the effects of light intensity and media on greenhouse-grown and in vitro grown cultures of
‘Redcoat’. Calli from in vitro grown cultures had very poor regeneration capacity. Both
greenhouse-grown and in vitro cultures formed callus and shoots when BA and 2, 4-D
were used at different concentrations. Severe browning occurred when fully expanded
leaves of greenhouse grown ‘Redcoat’ were used. Browning was caused by BA and NAA
and that it occurred even when young leaves were used. They suggested that the browning
would not have occurred in the presence of activated charcoal added to the medium.
However, activated charcoal will inhibit callus formation. They also saw a relationship
between hormone concentrations and explant polarity when tissue from the upper part of
the leaf regenerated at a higher frequency with higher concentrations of hormones than
those on the basal end exposed to lower concentrations of hormones.
Narender et al. [120] reported that Plant regeneration using immature leaf explants taken
from in vitro shoots and greenhouse grown plants of the strawberry cv. Redcoat. Both
types of explants formed callus and multiple shoots at various frequencies in the presence
of benzyladenine (BA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) at different
concentrations. Highest shoot regeneration (31%) occurred with the calli of greenhouse
leaf explants at 5 µM each of BA and 2,4-D in 24 weeks. Improved regeneration (50%)
with vigorous shoot proliferation was achieved in 10 weeks by transferring the 5 week old
green compact calli derived from greenhouse leaf explants to MS basal medium
supplemented with 10 µM BA and 1 µM 1-naphthaleneacetic acid (NAA) and incubating
the cultures at 16 h light (62.5 µE. m -2 s-l). The calli from in vitro leaf explants had very
poor shoot regeneration capacity. Seven other cultivars of strawberry also responded to this
regeneration protocol albeit at different frequencies. The protocol may be useful for
improvement of strawberry through somacional variation and selection of desirable traits.
Saifullah et al. [121] demonstrated that the potential of callus culture and regeneration was
evaluated in strawberry (Fragaria ananassa). The effect of different hormonal
combinations and explant sources was studied in- order -to produce maximum number of
plant in shortest possible time. Young leaves developing from glasshouse and in vitro
grown strawberry plants of cultivar Tango cultured on MS mineral salt. They observed that
the organic potential of explants from in vitro culture shoots compared with those taken
from glasshouse grown plants was significantly different for the sane media. The calli
induced from in vitro grown plant exhibited high regeneration as compared to those
induced from glasshouse grown plants. They also saw that maximum shoot regeneration
from in vitro was found on regeneration media 29 containing BAP/IBA 2.25/1.0 mg\l
whereas, glasshouse grown explants gave maximum shoots on medium 13 containing
BAP/NAA 2.25\1.0 mg\l. The poor shoot regeneration on leaf explant taken from in vitro
shoots may be due to a hormonal effect between NAA and IBA interaction caused by
accumulation of IBA in the leaf tissue.
Wojciech et al. [122] demonstrated that the growth in vitro cultures of strawberry
(Fragaria × ananassa Duch.) depending on different photoperiods. The investigated
photoperiods influenced growth of strawberry clones in vitro on Boxus media
supplemented with glucose. The application of 22/2 (d/n) photoperiod was not
advantageous as it did not improve shoot proliferation of both ‘Elsanta’ and ‘Senga
Sengana’ in vitro cultures, whereas stimulated the growth of callus at the explant base and
caused chlorosis of leaves when compared to control [16/4 (d/n)] .Photoperiod 4/2 (d/n)
significantly enhanced shoot proliferation of ‘Senga Sengana’ cultures. However, in
principle such reaction of ‘Elsanta’ cultures was not distinct as the significantly better
proliferation of shoots under 4/2 (d/n) photoperiod was only recorded in the third passage.
The leaf blades of both clones grown under 4/2 (d/n) cycle were significantly reduce. On
the other hand, the leaf petioles of ‘Elsanta’ were visibly elongated under mentioned
photoperiod.
2.5.3 Strawberry Anther Culture and Haploid Recovery
Anther culture involves the aseptic culture of immature anthers to generate fertile haploid
plants from microspores. The production of haploid plants through anther culture is widely
used for breeding purposes, as an alternative to the numerous cycles of inbreeding or
backcrossing usually needed to obtain pure lines in conventional breeding. Chromosome
doubling of haploids could result in immediate establishment of homozygosity for all loci.
The success achieved with anther culture has led to the development of microspore culture
to regenerate homozygous plants. Furthermore, isolated microspores are very attractive for
protoplast isolation and applications aiming at transformation as they are unicellular and
transgenic homozygous plants could be provided in a comparatively short time [39].
Haploid recovery in strawberry through aseptic anther culture was unsuccessful in some
early reports [123, 124].
Owen and Miller [125] obtained haploid plants from cultured anthers of ‘Chandler’,
‘Honeoye’ and ‘Redchief’ strawberries. The highest shoot regeneration across cultivars
(8%) was obtained when a semi-solid MS medium contained 2 mg/l IAA, 1 mg/l BA and
0.2 M glucose. Chromosome counts of root tip cells from ex vitro-grown plants confirmed
that haploid plants were obtained from all three cultivars.
Hamdouni et al.
[126] demonstrated that the achenes of two strawberry cultivars
'Chandler' and 'Tudla' when scarified and sterilized by H2SO4 (36N), showed a maximum
capacity of germination after a short time on MS inorganic salts (‘Chandler’ 94% and
‘Tudla’ 63%). On the contrary, the hydrogen peroxide treatments did appear to affect
neither the time nor the rate of germination. The culture on the same medium
supplemented with 3% sucrose of achenes cut and treated by H2SO4 1N, as well as
isolated embryos give very good results (97% and 100% for ‘Chandler’ and 74% and 85%
for ‘Tudla’, respectively for cut achenes and embryos), and shown the role played by
teguments in the dormancy of achenes and reduced the time to 50% of final germination to
only 3 days.
2.5.4 Strawberry Somatic Embryogenesis
Somatic embryogenesis research with strawberries is still in a preliminary stage and some
more efforts would be required to develop the technology [127].
Wang et al. [128] reported somatic embryogenesis from strawberry cotyledons on MS
medium supplemented with 22.6 ìM 2, 4-D, 2.2 ìM BA and 500 mg/l casein hydrolysate
where few of the embryogenic tissues developed into somatic embryos. Morphologically
normal plants were obtained from somatic embryos that were transferred to MS medium
containing 2.89 ìM GA3 or 2.22 ìM BA + 0.54 ìM NAA. Maintenance of the
embryogenic cultures was, however, unsuccessful.
Donnoli et al. [129] reported somatic embryogenesis in 8% of the embryogenic calli in
strawberry cultivar ‘Clea’ on MS medium supplemented with 4.88 ìM BA and 4.90 ìM
IBA.
2.5.5 Strawberry Somaclonal Variation
The variation in the regeneration capacity amongst different cultivars has also been
observed in other studies:
Strawberry plants spread vegetatively using runners and this enables them to be easily
transplanted and propagated as clones. Commercial production of strawberry using
micropropagation processes bears several risks. Plant off-types, i.e. non true-to-type and
genetically not identical to the mother plant, may be among the resulting plants. These
plants can simply the result of hardening errors and not arise from a change in the genetic
make up of the plant [130].
In vitro production of plants involves the application of plant growth regulator, such as
auxins, for process initiation. Nevertheless, these auxins are known to be associated with
genetic instability in plants, a phenomenon called somaclonal variation. Although,
somaclonal variation may be used as a source for variation to get superior clones, it could
be also a very serious problem in the plant tissue culture industry resulting in the
production of undesirable plant off - type [130].
However, through micro-propagation of strawberries (Fragaria x ananassa, Duch.),
several morphological abnormalities were detected as somaclonal variation. Changes
include leaf color variants and dwarf plants, among others [131]. Callus-derived “Redcoat”
strawberry plants differ significantly from standard runner plant for several vegetative
characteristics [132]. These variants pose a problem for production of uniform, true-to-type
plants.
Nehra et al. [133] found that two cultivars of strawberry responded differently to various
forms of in vitro propagation and in both cases variants were found in callus-derived
plantlets, but not those derived from meristems or via direct leaf regeneration. Some
studies have shown that modified characteristics are epigenetic and disappear over time
[134]. Numerous authors have reported that genetic changes including insertions,
deletions, point mutations and rearrangements occur during tissue culture [135], but few of
the phenotypic symptoms found are heritable [136, 137]. Most somaclonal variations occur
in plants regenerated from cultures that have undergone a differentiation phase.
Some studies indicating that a strong genetic component determines the success of
adventitious regeneration. Recently, a role has been suggested for endogenous H2O2 in the
process of bud primordia formation from strawberry calli [138]. Along this line, Yonghua
et al. [139] found that shoot regeneration was enhanced when explants were cultured under
red or green plastic films, and this was correlated with an increase in activity of antioxidant
enzymes and endogenous hormone concentration.
Presently, there are various methods available which can be used to detect and monitor
tissue culture-derived plants and cultivar identification. The most reliable methods are the
molecular marker techniques that identify the variance depending on the plant proteins,
which are expressed from defined regions of DNA, or DNA polymorphisms. RAPD
(random amplified polymorphic DNAs) is a powerful technique for identification of
genetic variation. It has the distinct advantage of being technically simple and quick to
perform, requiring only small amounts of DNA compared to restriction fragment length
polymorphism (RFLP) analysis. Strawberries (Fragaria × ananassa Duch.) have been
extensively analyzed using randomly generated markers for clone identification and
diversity studies [130].
Damiano et al. [140] studies about somaclonal variation and in vitro regeneration of
strawberry cultivar ‘Teodora’ and ‘Clea’, micropropagated on MS medium, they proved
that the variability induced by the in vitro culture has been shown at callus, cell level and
after regeneration from well organized tissues. They shown that petioles and laminas,
produced poor callus only, but the stipules are highly competent for organogenesis, and the
BAP alone is sufficient to induce regeneration, while 2, 4-D is strongly inducing callus
formation. Cell suspention and calli can be selected for deferent characterization of growth
carves and polymorphisms of the isoenzymes.
Polymorphisms also appear in acid-phosphatase, glutamate-dehydrogenase and peroxidase
of regenerated plants. The in vitro naturally occurring variability has been used for
selecting cell lines to be resistant Colletotrichum acutatum crude culture filtrates; the
selection effectiveness is shown to be dependent on the age of the fungus cultures and on
the dosage of filtration liquid. The regenerated plants show polymorphism at RAPDs
analysis. The sensitivity to the treatment with culture filtrate of C. acutatum was measured
with test of viability (fluorescein diacetate). The viability is varying according to the
dosage, the length of the incubation of fungus and the genotypes.
Adel 2007, [131] studied
the somaclonal variation in micro-propagated strawberry
detected at the molecular level.
Meristem tips of three Strawberry cultivars, namely
Chandler, Sweet Charlie and Gaviota were excised and cultured for 5 weeks on the starting
medium. Subculture was carried out for five weeks on shooting medium, and finally shoots
transferred to the rooting medium. The obtained plantlets were acclimated under
greenhouse condition in medium consisting of peat moss and sand (2:1). DNA extracted
from in vitro-derived plantlets and standard propagated plants were analyzed by RAPDPCR to detect possible drift in genetic stability of micro-propagated plants. He showed that
most of the obtained bands from in vitro-derived plantlets in all primers used with the three
cultivars were found to be present in the fingerprints of standard propagated plants,
demonstrating no variation in the pattern obtained with DNAs from the two sources of
strawberry plants. It is concluded that mass propagation via meristem tip culture is reliable
in producing genetically similar plants to the mother ones.
CHAPTER 3
Materials and Methods
3.1 Materials
3.1.1 Source of Explants
Mother plants of strawberry (Fragaria x ananassa Duch.) cv. Sweet Charlie grown in the
farm land of Bith Lahia city, Gaza Strip, Palestine, were used as source of explants during
this study. Runner tips of 1-2 cm long were taken from mother plants as that long was
suitable for sterilization procedures.
3.1.2 Chemicals
The chemicals that were used are listed in table
Table 4. A list of the chemicals used in this work.
Chemicals
Manufactures
Macro elements
 NH4NO3
 KNO3
 CaCl2.2H2O
 MgSO4.7H2O
 KH2PO4
 Na2EDTA
 FeSO4.7H2O.
Micro Elements
 H3BO3
 MnSO4.4H2O
 ZnSO4.7H2O
 KI
 Na2MoO4.2H2O
 CuSO4.5H20,
 CoCl2.6H2O
Organic Constituents
 Glycine - Myo-inositol - Nicotinic Acid
Pyridoxine HCl - Thiamine HCl
 Sucrose
 Agar
Hormones
 Benzylaminopurine (BA)
 Indolbutyric acid (IBA)
Detergents
NaOCl - Tween 20 - HgCl2 - Ethyl alcohol 70%
Suppluments
 Polyvenylpyridazone (PVPP)
 Activated charcoal (AC)
Another
 NaOH – HCl - KOH
Sigma Company
Sigma Company
Hi Media Company
Hi Media Company
Hi Media Company
Hi Media Company
Sigma Company
3.1.3 Equipments
The main equipments that were used are listed in table.
Table, 5. A list of the main equipments used in this work.
Instruments
Manufactures
 Growth chamber
Islamic Universty Lab.
 PH meter
Made in Germany.
 Autoclave
Henna (Made in Germany).
 Analytical balance
a-e ADAM PW 254
 Stirrer
Heidolph. MR 3001K
 Micropipette
Tomos Biotools Co., Ltd.
 Lux Meter
Made in Japan.
 Water Bath
Gemmy Industrial Corp YCW – 01
 Microfilter
Hi media Company
3.2 Methods
3.2.1 Surface Sterilization of Explants
Explants were rinsed under running tap water for 30 minutes. For surface sterilization,
explants were immersed in different concentrations (0.5, 1, 1.5, 2.0 and 2.5%) of sodium
hypochlorite solution containing two to three drops of Tween 20 per 100ml for 20 minutes.
The plant materials were then, rinsed three times with distilled water and were treated with
0.1% mercuric chloride solution for 10 minutes. The explants were then thoroughly
washed (4 - 5 washings) with sterilized distilled water to remove the traces of HgCl2. After
sterilization, explants were shortened to remove the surfaces of explants and meristems of
3-5mm long were isolated as final explants. These explants were cultured with good
contact on the surface of the medium.
3.2.2 Culture Medium
The media was formulated in the lab according to the composition of Murashige and
Skoog [4] referred as MS medium was selected as the optimal culture medium including
(0.2%) PVPP.
Stock solutions of generally 4x macroelements, 1000x for microelements, 100x for the
organic constituents were prepared. These stock solutions were stored in a freeze chest at
o
4 C and were mixed in desired proportions only before use. None of the stock solutions
were stored for more than 15 days.
Table 6: Murashige and Skoog’s Medium (1962) [4] Constituents Amount (mg/l).
No.
1
2
3
4
5
6
7
Macro Elements
Conc. mg/L
NH4NO3
KNO3
CaCl2.2H2O
MgSO4.7H20
KH2PO4
Na2EDTA
FeSO4.7H2O
1650
1900
440
370
170
37.31
27.81
No.
Micro Elements
Conc. mg/L
1
2
3
4
5
6
7
H3BO3
MnSO4.4H2O
ZnSO4.7H2O
KI
Na2MoO4.2H2O
CuSO4.5H20
CoCl2.6H2O
6.2
22.3
8.6
0.33
0.25
0.025
0.025
No.
1
2
3
4
5
6
7
Organic Constituents
Conc. mg/L
Glycine
Myo-inositol
Nicotinic Acid
Pyridoxine HCl
Thiamine HCl
Sucrose
Agar
2.0
100
0.5
0.5
0.1
30.000
0.6%
The reagents used were of Analytical Reagent Grade. Each salt was dissolved separately
one after one to avoid precipitation.
3.2.3 Sterilization of the Nutrient Media
Following preparation of the medium and prior to addition of agar, the pH was adjusted to
5.8. The medium was poured into culture jars of size 350ml where each jar contained 30
o
ml of the medium and capped with plastic caps. The culture jars were autoclaved at 121 C
and 1.2 kg/cm2 air pressure for 20 minutes.
3.3 Strawberry Micropropagation
3.3.1 Effect of Pretreatments on Explant Browning
To study the effect of various pretreatments on explant browning and survival percentage,
different sets of sterilized explants were dipped in three different concentrations of
antioxidant solution (1, 1.5 and 2% PVPP) and another set dipped in sterilized distilled
water without antioxidant solution as a control.
Meristem tips were cultured on hormone-free MS solid medium to establish clean and
healthy materials for next experiments.
Cultures were kept for chilling treatment at 4 oC for 10 days and then incubated for 4
weeks in the growth room.
All cultures of this study were incubated at 25 oC ± 2 day and night. Light was provided by
fluorescent tubes giving an intensity of 1500 lux at the level of culture vessels for 16 hours
per day.
3.3.2 Multiplication Stage
Meristem - derived plantlets were multiplied by sequential subculturing into fresh
medium in 300 mL jars each with 30 mL of culture medium supplemented with 1.0 mg/L
BA hormone and closed with plastic caps. At this stage of culture, the shoots proliferated
rapidly within one month. Two subcultures were then conducted.
3.3.3 Rooting Stage
3.3.3.1 Effect of Different Strengths of MS Medium
Shoots of strawberry obtained from multiplication stage were cultured on different
strengths of MS, 1/8, 1/4, 1/2, and full strength of MS medium. All treatments of this set of
experiment contained hormone free media. After 4 weeks, data were recorded as, root
number/plant, root length, shoot length and fresh / plant.
3.3.3.2 Effect of Different Concentrations of IBA
Shoots of strawberry obtained from multiplication stage were cultured on different
concentration of IBA, 0.5, 1.0, and 1.5 mg/L. After 4 weeks, data were recorded as, root
number/plant, root length and shoot length and fresh /plant.
3.3.3.3 Effect of Different Concentrations of Activated Charcoal
Shoots of strawberry obtained from multiplication stage were cultured on different
concentration of activated charcoal, 0.2, 0.5 and 1.0 mg/L. All treatments of this
experiment contained hormone free media. After 4 weeks, data were recorded as, root
number/plant, root length and shoot length and fresh /plant.
3.4 Statistical Analysis
All experiments were repeated twice and the represented data were averages. Results of
these experiments were analyzed by analysis of variance (ANOVA).
CHAPTER 4
Results and Discussions
4.1 Source of Explant
The strawberry plant used in this investigation were chosen on the basis of being the most
commercially successful varieties currently grown in the Gaza strip, In addition cv. Sweet
Charlie was selected for two reasons: it is the most economically important cultivar
currently grown in Israel and Gaza strip and it has many characteristics that could be
improved in a genetic modification programme. It is generally agreed that plants
regenerated from shoot tips or buds are genetically stable and free from somaclonal
variations associated with plants differentiated from callus [141].
In vitro culture of strawberry greatly affects the quality of material obtained and its field
performance [94]. Generally, cultures are most easily established from explants harvested
from the active growth of the mother plant. The best time for collecting explants is usually
when stock plants are producing new vigorous shoots (i.e. in temperate regions, in spring
to early summer), but many exceptions have been observed. The optimum time for
collecting explants may depend on the kind of culture, on the particular genotype to be
used, on the relative amounts of contamination, and browning of the explant occurring at
each season [19]. However, in our experiment it was observed that establishment stage in
vitro culture of strawberry depends on several factors such as: the quality of material
obtained, shoot collecting season of explants, contamination treatment without any effect
on the explants and reduction browning due to reaction between the phenol exudation
secretion from the explants and some chemical reagent such as sodium hypochlorite.
4.2 Surface Sterilization of Explants
One of the main problems encountered with in vitro propagation was the massive bacterial
contamination at the initiation and multiplication stages. After transfer of the bud to solid
sterile medium, a whitish exudate of bacteria was observed around the base of the explant
after 2-3 d. The problem was complicated further by the latent nature of the contaminants
[142].
In general, it is accepted that contamination of plant tissue cultures can be caused mainly
by insufficient aseptic techniques during manipulations, incomplete surface sterilization of
the explant and endogenous microflora present in the explant.
We think that the most difficult stage in tissue culture is a contamination and browning.
These two problems are combined with each other. To decrease the browning, we should
be careful during the surface sterilization process of the explant. However, none of the
reports mentioned the percentage of clean cultures obtained nor the occurrence of
phytotoxic effects. This result agrees with Moutia et al. [57] he reported that disinfection
with 2.7% sodium hypochlorite (pH 6) proved to be most phytotoxic, resulting in 100%
necrosis and death of buds.
Sodium hypochlorite and mercuric chloride have been used by many authors for surface
sterilization of strawberry explants, Passey et al. 2003 used (0.525% sodium hypochlorite)
Bhatt and Dhar [143] used (0.05% HgCL2) none of them have dealt with the problems of
bacterial contaminants and browning occurring during culture.
In the experiments reported here, both chemicals gave good reduction of bacterial
contaminants and gave the maximum aseptic cultures and maximum explant browning in
cv. Sweet Charlie except the treatment with 1.5% Sodium hypochlorite which gave the
best results, especially, when the explant treatment with 1 and 2% PVPP for 1-2 h before
culture, the browning percentage were 80 and 40% respectively, and survival percentage
were 20 and 60% respectively in August season (Table 7).
Moutio et al. [57] explain that none of the surface-sterilizing agents used achieved total
elimination of bacterial contaminants when using sodium hypochlorite alone and mercuric
chloride alone, and they demonstrated that the disinfection with 1% mercuric chloride gave
56.3% clean cultures as opposed to 46.8% with 2.7% sodium hypochlorite. However, in
our experiment we think that the combination with two treatments increase the reduction of
contamination.
According to these notes in our experiment is acceptable that we could reach the suitable
concentrations in sterilization and browning methods. By this way we have got survival
explants.
While the same experiment was applied on the explants but was collected in October
season and from other field, we observed that all the samples were failed to reduce
explants contamination. We believed that these results depend on the selection of the
material in the field and choice a good farm land which used better bacterial and fungal
treatment before and after culture, or that the shoot collection season proved to play
important role in reducing contamination. Boxus (1999) [144] reported some ways to
select the best mother plants in the field. The very young runner tips are the best plant
material, and must be collected in a fruit production field in June, before the runners have
formed leaves and roots. This very young material has fewer hairs surrounding the
meristematic zone and results in little contamination. Swartz et al. [95] noted that the
contamination problems occurred with cultures initiated during late Summer and Autumn,
or some laboratories keep the mother plants in screen houses for several years, and always
use the same plants for the meristem excision.
Persistence of contamination after strong disinfection and the sudden appearance of visible
bacterial growth during later in vitro stages led to the hypothesis of endogenous, latent
bacteria in the explants. Such observations have also been suggested by many authors
working with in vitro culture of ornamental, fruit and crop species. Many bacteria found in
old plant tissue cultures are known not to produce symptoms on the plant or visible growth
on the propagation medium in vitro and these are usually described as being latent,
endophytic or endogenous. The growth of these bacteria is often limited and multiplication
occurs as a result of changes induced by the plant. Such bacteria are not detected by visual
assessment of plant cultures and are propagated together with the plant material.
Multiplication rates of plant cultures contaminated with such bacteria were, however,
shown to decrease and in some cases, plants were killed by the phytotoxic metabolites
produced by the bacteria [145].
This high percentage of endogenous saprophytes could be explained by the age of the buds
used. It is generally accepted that ageing of tissue is accompanied by an increase in
microbial infection. The West Indies Central Sugar Cane Breeding Station in Barbados
reported that older and mature buds of sugarcane were more susceptible to infection than
younger ones [146].
Table 7. Surface sterilization treatments for the elimination of bacterial
contaminants in in vitro bud culture of strawberry cv. Sweet Charlie (The
treatment condition as mentioned in section 3.3.1)
Sterilizing agent
Percentage of explant
contamination
Sodium hypochlorite 1.0%
Sodium hypochlorite 1.5%
Sodium hypochlorite 2.0%
Sodium hypochlorite 2.5%
0
0
0
0
Percentage
of explant
browning
(1% PVPP)
100
80
100
100
Percentage
of explant
browning
(2% PVPP)
100
40
100
100
Another investigation, in our study the shoot collection season plays an important role in
reduction of browning and bacterial contaminants. Indra et al. [147] reported that shoot
collection season proved to play important role in reducing percent browning and inducing
bud break. Explants collected during winter (November - December) gave the maximum
response as measured by percentage of explants with axillary bud break in vitro, and they
observed that seasonal changes greatly influence explant establishment and cultures could
not establish in rainy season due to heavy fungal and bacterial contamination.
The effect of tissue culture on leaf formation was studied by Zebrowska et al. [148] who
revealed that number of leaves per microplant in the Autumn after planting and in the next
year of cultivation was significantly higher in comparison to the standard propagate
seedlings.
4.3 Effect of Pretreatment on Explant Browning:
In the experiments reported here, to study the effect of various pretreatments on explant
browning and survival percentage. The results of the present study demonstrated that the
concentration of PVPP was beneficial to reduce exudates and inhibit the browning in the
October season, the highest percentage were observed in the control and 2% PVPP
compared with 1.5% and 1% PVPP respectively (Table 8 ) and low survival percentage for
all treatments, these results are considered abnormal.
Table 8. The effect of PVPP in the browning inhibition.
Time
1% PVPP
3 clear
7 moderate
0 high
2 clear
12 days
7 moderate
1 high
Clear = (without browning),
6 days
1.5% PVPP
2% PVPP
Control
5 clear
7 clear
10 clear
3 moderate
2 moderate
0 moderate
2 high
1 high
0 high
4 clear
7 clear
10 clear
4 moderate
2 moderate
0 moderate
2 high
1 high
0 high
moderate = (moderate browning), high = (high browning)
However, the results can be explained on the basis that PVPP 0.2g/100ml when added into
medium and subjecting cultures for ten days to cold treatment before transfer the explants
to growth chamber was found most effective resulting in 100% browning inhibition in
control. This finding is consistent with the fact that addition of PVPP into the media alone
able to eliminate and prevent the browning. It is known that cell death is correlated with
elevated levels of peroxides during tissue browning; in addition there are many factors able
to induce browning after the explants treatment such as temperature and light intensity
through the experiment.
In some plants higher peroxidase activity is induced by mechanical injury, environmental
stress, H2O2, and polyphenols [149]. We speculate that wounding derived from these stress
responses may be associated with the ability of peroxidase to detoxify oxidative damage
and cause cell death, as well as subsequent tissue browning. When antioxidants were added
to the plant tissue-culture medium, they reacted with polyphenols and decreased the
concentration of harmful compounds. Antioxidants reduced the accumulation of
peroxidase in strawberry tissue and protected cells from oxidative damage. Antioxidants
also reduced the inhibition of polyphenols to cell growth and shoot formation; therefore,
the frequencies of runner tips induction, shoot differentiation, and rooting were increased.
The results from this study indicate the browning phenomenon in strawberry runner tissue
can be greatly reduced by addition of 0.2g/100ml PVPP into the medium culture. Also,
incubation in the cold at 4oC for the first 10 days may arrest the rate of tissue browning by
slowing the enzymatic activity responsible for tissue oxidation (Table 7). Frequent
subculturing to fresh medium may also assist so that toxic phenolic compounds do not
hinder the activity of plant growth regulators on tissues. Madany et al. [111] show that
addition of 0.2 % activated charcoal was better than 2% PVPP to eliminate inhibitory
substances from in vitro cultures of (Fragaria x ananassa Duch.) cv. Camorosa and Selva.
PVPP added in the medium was also found effective in explant browning prevention from
earlier reports on guava and Cleistanthus collinus [147].
Nehra et al. [150] Observed that the explants taken from fully expanded, mature
greenhouse leaves of strawberry cv. Redcoat exhibit severe browning. The combination of
BA and NAA caused browning even in the explants obtained from young leaves. Explants
browning caused by this treatment could be effectively controlled by the addition of
activated charcoal to the medium.
Indra et al. [147] explained that in the spring season maximum explants died due to
phenolics. This is perhaps due to higher phenolic content in growing shoots.
Media containing only nitrogen as nitrate are used for the shoot culture of some plants, for
example strawberry (Boxus, 1974) [91], which can be cultured with 10.9 mM NO3 alone;
supplementing the medium with 6 mM NH4+ causes phytotoxicity. However, shoot
cultures of strawberry grown without NH4+ can become chlorotic: adding a small amount
of NH4NO3 (or another source of reduced nitrogen) to the medium at the last proliferation
stage, or to the rooting medium, may then give more fully developed plants with green
leaves. On some occasions it is necessary to eliminate or reduce NH4+ from the medium for
shoot cultures to prevent hyperhydricity [19].
Meria et al. [151] observed in preliminary testes that using MS basal salts instead of WPM
basal salts in strawberry culture ( Arbutus unedo L.) resulted in extensive oxidative
browning and failure of explants after culturing for 3 weeks, this browning was due to a
high concentration of salts or, in particular, due to high NH4NO3 concentration in MS
medium.
We believe that incubation the culture in the cold at 4oC for the first 10 days preventing
high nitrogen content in the MS medium and alleviated the problem of browning and
toxicity. This finding further supports the importance of this treatment in promoting
organogenesis in strawberry. In that concern, Passy et al. [115] focused that pre-chilling of
stock material at
4oC enhanced subsequent shoot regeneration.
Some laboratories prefer MS medium to Knop medium as macronutrients during the
rooting phase. Indeed, on this medium, the plantlets will grow faster. However, these MS
plantlets are softer and also more susceptible during the weaning phase [146].
After transfer the samples to the growth room the runner tips starts swilling and about two
weeks later, the excised meristem tips have developed into a rosette of leaflets, and has
developed to a size of a few millimeters (Figure 3. A, B).
Within 3 - 4 weeks after this excision, the shoots reach to 2-3 centimeters (Figure 4. A, B),
transfer to MS propagation media supplemented with 1mg/l BA hormones.
B
A
Figure3 :(A) Swilling of runner tips explants of cv. Sweet Charlie on MS medium free
hormone after 8 days of culture.(B) Development of bud from runner tips explants
(arrow) after 15 days of culture.
A
B
Figure 4. (A). Runner tips explants of strawberry in MS media free hormone after 4
weeks culture the leaf reach to 2-3 cm. (B). Explant after 6 weeks in MS media
supplemented with 1.0mg/l BA.
Within 3 - 4 weeks after this transfer, two or three new buds appeared at the base of the
petioles of the oldest leaves. These young axillary buds grow very quickly and, in turn,
produce new axillary buds. After 6 – 8 weeks the axillary buds invade the base of the
petioles of the oldest leaves (Figure 5). The initial plantlet is transformed into a cluster of
buds, these clusters may contain more than 15 – 25 small buds. No roots are present on these
buds.
A cluster of buds
Figure 5: Explant in multiplication media after 8 weeks, the axillary buds invades the
base of the petioles of the oldest leaves (first division).
4.4. Multiplication Stage
At this stage, the clusters0of buds may be aseptically separated and transferred onto a fresh
medium to continue the clonal propagation. Every 6 – 8 weeks, small portion of the
isolated buds produced high number of new axillary buds (Figure 6).
During the second division of the clusters, only a small fraction of the isolated buds is
transferred to proliferation medium to continue the propagation.
Axillary buds
Figure 6: Induction of multiple shoots from Runner explants on MS medium
supplemented with 1.0 mg/L BAP, after 12 weeks of culture.
In our experiments the multiplication stage play an important role in micropropagation
process, our results indicated that medium contained 1.0 mg/l BA alone proved to be the
best for shoot formation, shoot number and number of buds. The results are in agreement
with those of Emara (2008). [36] who reported that BA alone at 1.0 mg/l significantly
showed the highest record of shoot number followed by the treatment contained 1.0 mg/l
BA and 0.1 IBA that a significant similar response. Boxus (1999). [146] revealed that
when shoot tips of strawberry were cultured on a medium supplemented with 0.5 mg dm-3
BA, 0.1 mg dm-3 GA3 and 6.4 g dm-3 agar, shoots were multiplied, while Marcotrigiano et
al. [152] found that BA at 0.3 mg/l was as effective for shoot proliferation as 1 and 3 mg/l.
The importance of BA, in strawberry shoot formation, was also reported by Lal et al. [153]
who found that the maximum number of shoots per explant was observed in MS medium
supplemented with BAP at 4.0 mg/l.
Benzyladinine seems to be important in controlling proliferation of strawberry shoots.
Marcotrigiano et al. [152] observed on a large number of strawberry cultivars that BA
concentrations in the range 1.3-13.3 mM, did not produce significant differences in
multiplication rate; in contrast, Simpson et al. [154] found clear differences among
genotypes in their BA requirements for optimum shoot proliferation. In our case, optimum
BA level for cv. Sweet charlie was 1.0 mg/l gave better results, Lopes et al. [94] indicated
that the use of low BA concentrations on strawberry has been recommended by several
authors, since it decreases the risk of phenotypic abnormalities after the field establishment
of micropropagated plants [154].
In that concern, the use of 0.5 mg/l BA and 0.5 mg/l IBA was reported for shoot
proliferation of strawberry. Singh and Pandey [155] reported that half strength MS medium
supplemented with 1 mg / l BA and 1 mg/l IBA produced the highest number of shoots (13
per explant). Kaushal et al. [156] found that callus of strawberry differentiated into shoots
after transferring to MS medium supplemented with 0.5 Kin + 2 mg/l BA + 0.25 mg/l
NAA. Well developed shoots were transferred to multiplication medium containing 0.5
mg/l Kin + 0.5 mg/l BA and 1.0 mg/l GA3. Weifeng et al. [157] revealed that Kin used
together with BA for adventitious bud induction gave better results than Kin alone, but,
Waithaka et al. [158 ] revealed that axillary dormancy was released by including a large
level (50 µM) of Kin in the culture medium.
The results can be explained on the basis that different plants and even different organs of
the same plant are characterized not only by their unique intrinsic biochemical make-up
but also by the sensitivity of the endogenously supplied chemical stimuli.
Economically, the division within isolated buds is not very easy; it takes a lot of time.
Therefore, after one or two transfers on proliferation medium, the cluster are now divided
more roughly into four to five small tufts of buds. To maintain a high proliferation rate, the
transfers must be done immediately after the appearance of the first roots. In this way, the
proliferation rate is about four- to fivefold per month, i.e., several million from one
meristem in only one year [146].
Lopes et al. [94] explained that the number of subcultures (3-8) did not affect productivity;
in accordance with another observation they pointed out that problems arise when using
material with over 10 subcultures.
4.5. Rooting Stage
4.5.1. Effect of Different Strengths of MS Medium
The results on the effect of MS strengths on root number and root length indicated that,
highest response was obtained with full MS followed by 1/2
MS, but 1/4 and 1/8 MS
came in the second order (Figure 7). In case of full MS the root number, root length and
shoot elongate was high response, but 1/2 MS the root number was high but root length
and shoot elongate was less than full MS.
Lower responses were obtained with the 1/4 and 1/8 MS with respect to root number and
root length, but the shoots remained too short for rooting.
Figure 7: The effect of different strengths of MS, 1/8, 1/4, 1/2, and full strength of
MS medium on rooting response after 4 weeks, highest response was obtained with
full MS followed by 1/2 MS, but 1/4 and 1/8 MS came in the second order.
4.5.2. Effect of Different Concentrations of Activated Charcoal
The effect of different concentration of AC on root number and root length revealed that
the highest response was obtained with 0.5 mg/L, similar to full MS, all other
concentration showed lower effect in elongation of both the roots and shoots.
4.5.3. Effect of Different Concentrations of IBA
Regarding the effect of IBA on the root response, the results indicated that the IBA with
(0.5, 1.0, and 1.5 mg/L) showed the lowest roots response compared to all other treatments.
These findings are somewhat similar to those previously reported by Emara (2008). [36]
who studied the effect of MS strengths on root length, who reveal that the highest
significant record was obtained with full MS, all other strengths showed lower effect.
Induction of roots at the base of in vitro grown shoots is essential and indispensable step to
establish tissue culture derived plantlets to the soil. The most effective auxins for rooting
are IBA and NAA [18].
In that concern, some investigations did not recommend full MS for strawberry rooting,
Kaushal et al. [156] found that rooting of strawberry was done in MS half strength with 1.0
mg/l IBA and 0.2 mg/l activated charcoal. The same strength was recommended for
strawberry rooting by Yonghua et al. [139] who reported that half strength MS containing
1.0 mg/l AgNO3 was optimum medium for rooting. Kikas et al. [96] found that high salt
concentration of MS promoted active proliferation but the shoots remained too short for
rooting, and with low salt concentration of MS, the proliferation rate was lower but shoots
elongated enough. Gautam et al. [159] indicated that the highest root induction frequency
obtained was 95.23% on 1/4 MS medium with IBA at 1.0 mg/l and charcoal (200 mg/l).
However, Lal et al. [153] found that maximum rooting was obtained in both full and half
strength MS medium supplemented with IBA at 1.0 mg/l. Mereti et al. [160] found that the
highest percentages of rooting were achieved in MS medium contained 10 µM IBA (92%)
and 10 µM IAA (82%).
Passey et al. [115] indicated that the high levels of regeneration obtained from leaf discs
(100% with Calypso and Tango), they placed the shoots on rooting media R13 containing
3.0 mg/L IBA for 3 days under the growth conditions and the shoots were then transferred
to root elongation media R37 contains half-strength MS salts.
Boxus (1999), [146] revealed that when shoots tips of strawberry were cultured on a
medium supplemented with 0.5 mg \L BA, 0.1mg \L and 6.4g \L agar, shoots were
multiplied and subsequently rooted in vitro on the same medium without BA and GA3.
In that concern, Kikas et al. [96] found that high salt concentration of MS promoted active
proliferation, but the shoots remained too short during rooting period, but with low salt
concentration of MS, the proliferation rate was lower but shoots elongated enough.
Damiano et al. [140] found that the addition of 1 or 2 g of activated charcoal per liter to
MS medium promotes elongation of both the shoots and roots of strawberry.
Nissen & Sutter [87] found that increasing the concentration of auxin promoted rooting
without callusing, due to an increasing concentration of activated charcoal, which had an
effective role in absorption of some of the medium components.
Root formation and growth were better in BA-free medium containing activated charcoal
than in the medium without activated charcoal. Similar results were observed in bulb
formation. This could be due to activated charcoal regulating internal physiological
processes. It is also possible that a substance(s), which antagonizes cytokinin activity, was
adsorbed by activated charcoal [83]. Other research has shown that activated charcoal had
an effective role in absorption of unknown components, which were produced through
chemical processes within the media. Sometimes these unknown components played the
role of growth inhibitor at morphogenesis stages. In addition, the activated charcoal, which
eliminated light and provided a reasonable physical environment for the rhizosphere,
prevents undesirable callusing and helps rooting [87].
The poor root stimulation in our experiment may possibly be due to a hormonal imbalance
caused by accumulation of auxin in leaf tissues. Contrary to our observation, in other
genotypes of strawberry (Borkowska, 2001) [161] who studied the micropropagation of
strawberry cultivars Senga sengana, Kent and Kama were rooted by a medium
supplemented with 1.0 mg/L IBA. These possibility are somewhat similar to those
previously reported by Nehra et al. [134] whereby it was noted that the calli from in vitro
strawberry cv. Redcoat leaf explants had very poor shoot regeneration because the
hormonal imbalance due to maintained the explant on a high cytokinin medium prior to
explant preparation.
In our results, we think that different concentrations of IBA were necessary to optimize
roots regeneration; these results are similar to those obtained in various other studies ([103,
162] for strawberry). Those researchers found that different concentrations of growth
regulators were necessary to optimize regeneration from both types of explants.
Sometimes the organic potential of explants from in vitro culture shoots compared with
those taken from glasshouse grown plants was significantly different for the same media.
Saifullah et al. [121] observed that maximum shoots regeneration from in vitro was found
on regeneration media containing BAP/IBA 2.25/1.0 mg/l whereas, glasshouse grown
explants gave maximum shoots on medium containing BAP/NAA 2.25/0.1 mg/l. They
explained that the poor shoots regeneration on leaf explant taken from in vitro shoots may
possibly be due to a hormonal effect between NAA and IBA interaction caused by
accumulation of IBA in the leaf tissue.
It is notable that using a full strength MS media free hormone on establishment and rooting
stage of strawberry give us optimal results. We think that the hormones which present in
the strawberry plant play an important role in shoot and roots stimulation. These
possibilities are somewhat similar to those previously reported by Jammaly et al. [37]
whereby it was noted that the six cytokinines and two auxins were isolated and identified
in axillary shoots in strawberry. And they observed that Benzyladenine and riboside were
the major cytokinines whereas the free IAA and its conjugate IAA aspartate showed higher
concentrations in axillary shoots that means the amount of hormones inside the strawberry
tissues gave optimal growth conditions in shooting and rooting response.
Actually, we had hoped to achieve this research as completely, but unfortunately the Israeli
air force bombed the lab building at the Islamic university in 27-12-2008. Thus, this is
caused demolishing the building completely as well as the special lab of tissue culture. As
a result, there is a problem to repeat the experiment or to complete it. Even though, this
action happened, but we would reach to rooting stage which is considered as a progress
stage in this research. Finally, in this stage we wrote down data visually, but we couldn’t
take data and measurements which prove experiments procedures which lead us to the
statistic analysis.
CHAPTER 5
CONCLUSION AND RECOMMENDATIONS
5.1 CONCLUSION
This study illustrates a successful micropropagation system for strawberry cv. Sweet
Charly plants and examined the application of micropropagation protocols to assist the
production of strawberry mother plants with high quality and resistance to common fungal
and bacterial diseases. For in vitro propagation of strawberry cv. Sweet Charlie, Key
factors were investigated in this study include sterilization technique to establish
contamination free culture, control of phenolic exudates in explant preparation. The
selection of appropriate growth regulator levels to achieve successful in vitro regeneration
and the selection of appropriate growth regulator levels and some supplemented levels to
achieve the optimal rooting.
One of the most commonly encountered problems in in vitro culture establishment is the
contamination, the maximum aseptic cultures was obtained when the runner tips were
immersed in sodium hypochlorite solution (1.5%) containing two to three drops of ‘Tween
20’ per 100ml for 20 minutes and then in 0.1 % HgCl2 for 10 min gave the maximum
aseptic cultures and maximum explant browning in except the treatment with 1.5%
Sodium hypochlorite which gave the better results, especially, when the explant treatment
with 1% and 2% PVPP for 1-2 h before culture, the browning percentage were 80 and 40%
respectively, and survival percentage were 20 and 60% respectively in August season.
However, in October season all the samples were affected with contamination.
In spite of the low number of clean cultures obtained, micropropagation still remains the
best alternative for the rapid bulking of promising varieties whilst maintaining genetic
stability of the clone.
Further reduction in bacterial contamination can be achieved by growing mother plants,
ideally originating from tissue culture or from nurseries and in a dry, clean environment,
preferably a glasshouse. Overhead watering should be avoided and routine pesticide
treatment and fertilization should be practiced, and can be obtained in specific season.
The apical bud is defined as the bud located immediately below the apical meristem and
enclosed within the leaf sheath. Selecting these young tissues makes it possible to reduce
infection since the apical zone displays better aseptic conditions because of the reduced
size of the explant and the small area exposed to the external environment. Taylor and
Dukic [163] reported that when apical buds of sugarcane were used, contamination with
saprophytic micro-organisms was less (20% to 40%) than in axillary buds (>90%). Since
these buds are morphologically less developed than axillary buds, it is less likely that the
vascular tissue or bud scales are colonized by saprophytic fungi, yeast and bacteria.
This study found that tissues of experimental plant were high in phenolic compounds. The
oxidation of tissues was severe and proved deleterious to all tissues in the initial stages of
explant preparation. So, for reduction of phenolic compounds explants were pre-soaked in
antioxidant solution of 1% or 2% PVPP for 1-2 h prior to inoculation with addition of
(0.2g/100ml) PVPP in the medium and subjecting cultures cv. Sweet charly during the
initial ten days in cold treatment before transfer the explants to growth chamber. The
effect of the treatments outlined above proved beneficial to explant survival in vitro of
strawberry cv. Sweet Charlie runner tips.
To study the effect of various pretreatments on explant browning and survival percentage,
the highest percentage were observed in the control and 2% PVPP compared with 1.5%
and 1% PVPP respectively, survival percentage were low.
For successful explant establishment, the runner tips apices were cultured for 4 weeks on
MS basal medium free hormone, the runner tips starts swilling and about two weeks later,
the excised meristem tips have developed into a rosette of leaflets.
For successful explant multiplication, within 3 - 4 weeks after this excision, transfer to MS
propagation media supplemented with 1mg/l BA hormone, two or three new buds will
appear at the base of the petioles of the oldest leaves (within 3 - 4 weeks after this
transfer). After 6 – 8 weeks the axillary buds invade the base of the petioles of the oldest
leaves.
For successful rooted plantlets, the highest response was obtained when transfer the buds
to the MS medium full strength and to MS medium supplemented with activated charcoal
( 0.5 g/l), all two media without hormone.
Present investigations have thus shown that the economically valuable strawberry cv.
sweet charly could be regenerated in vitro via organogenesis. The protocol developed will
be useful for rapid in vitro propagation of the species and also for the subsequent genetic
manipulation studies.
5.2 Recommendations
Although, the tissue culture is new tool for plant propagation in our country, it is used in
the developed countries many years ago for its economical importance. To establish this
method on true basics must follow the following:
1. Establishing a special lab concerning with tissue culture equipments which comprise of
success of experiments coming true.
2. Establishing a special greenhouse concerning with tissue culture that is treated by the
true ways to facilitate sterilization methods and avoiding contamination problems.
3. The use of tissue culture technique in a comprehensive way including the importance
plants in our country, such as potatoes and tomatoes etc…
4. The use of various methods with tissue culture through out the continuing experiments,
we need a help from people those, who are specialized in this method, for example, the
culture by use variety organogenesis, callus induction and embryogenesis.
5. The tissue culture methods must support different techniques for the genetic analysis
unit (Genotyping) by using molecular marker to recognized and sure that seedling are
true to type and without somaclonal variation.
For strawberry tissue culture planting in this research we recommend the following:
1. Continuing experiments procedure to reach to the final stage which we couldn’t do it
and that is seedling production able to acclimatization without side environment and
without any troubles.
2. Concentrating on writing down notes culture procedures step by step to reach for
optimum ways in doing experiment and saving a lot of efforts, costs and avoide the
negative results.
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